Dynamic genomic changes in methotrexate-resistant human cancer cell lines beyond DHFR amplification suggest potential new targets for preventing drug resistance.

BACKGROUND: Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood. METHODS: We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance. RESULTS: The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance. CONCLUSION: The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.

Astrocyte elevated gene-1 mediates glycolysis and tumorigenesis in colorectal carcinoma cells via AMPK signaling.

To investigate the role of AEG-1 in glycolysis and tumorigenesis, we construct myc-AEG-1 expression vector and demonstrate a novel mechanism that AEG-1 may increase the activity of AMPK by Thr172 phosphorylation. The higher expression levels of AEG-1 in colorectal carcinoma cells were found but showed significant difference in different cell lines. To study the role of AEG-1 in colorectal cells, myc-AEG-1 vector was constructed and transfected into NCM460 colonic epithelial cells. We observed consistent increasing of glucose consumption and lactate production, typical features of anaerobic glycolysis, suggesting that AEG-1 may promote anaerobic glycolysis. Moreover, we noted that AMPK phosphorylation at Thr172 as well as pPFK2 (Ser466) was increased in NCM460 cells overexpressing AEG-1. Compound C may block AMPK and PFK2 phosphorylation in both control and AEG-1-overexpressed cells and decrease the glucose consumption and lactate production. The present findings indicated that reduced AEG-1 protein levels by RNAi may decrease the glucose consumption and lactate production in HCT116 colorectal carcinoma cells. The present identified AEG-1/AMPK/PFK2 glycolysis cascade may be essential to cell proliferation and tumor growth. The present results may provide us with a mechanistic insight into novel targets controlled by AEG-1, and the components in the AEG-1/AMPK/PFK2 glycolysis process may be targeted for the clinical treatment of cancer.

[Overexpression of p-Stat3 and Mcl-1, and their correlation with differentiation and apoptotic resistance in esophageal squamous cell carcinoma].

OBJECTIVE: To detect the expression of phosphorylated-signal transducer and activator of transcription 3 (p-Stat3) and myeloid leukemia-1 (Mcl-1) as well as their correlation, and to investigate the functional role of Stat3 and Mcl-1 in the pathogenesis of esophageal squamous cell carcinoma (ESCC). METHODS: Stat3 activity in ESCC cells was inhibited with JAK/Stat3 inhibitors (AG490 or JSI-124). Specific siRNA was used to inhibit the Stat3 expression. Cell apoptosis was detected by flow cytometry. Expression of Mcl-1 protein was determined by Western blotting. Expression of phospho-Stat3 (Tyr705) and myeloid leukemia-1 (Mcl-1) proteins in ESCC tissues was detected by tissue microarray and immunohistochemistry. The relationship between p-Stat3 or Mcl-1 aberrant expression and clinicopatholohical features of ESCC was analyzed. The correlation of their expression was also analyzed. RESULTS: Suppression of the Stat3 signaling activation in ESCC cells led to marked apoptosis, and dramatic reduction of Mcl-1 protein. The positive rate of phospho-Stat3 (Tyr705) expression was 45.0% in 50/111 of the ESCC tissue samples. The lower the degree of tumor differentiation, the higher the positive rate of phospho-Stat3 (Tyr705), showing a significant difference (P = 0.018). The positive rate of Mcl-1 protein expression was 72.1% (80/111), and the lower the degree of tumor differentiation was, the higher there was the positive rate of Mcl-1, with a significant difference (P = 0.026). There was a positive correlation between the expressions of p-Stat3 and Mcl-1 proteins (P = 0.012). CONCLUSIONS: In a subset of ESCC tissues, p-Stat3 (Tyr705) and Mcl-1 are overexpressed and positively correlated with each other, and both are correlated with tumor differentiation. Persistent activation of Stat3 contributes to apoptotic resistance in ESCC cells, and may be at least partly mediated through upregulation of Mcl-1.

Characterization of gene rearrangements resulted from genomic structural aberrations in human esophageal squamous cell carcinoma KYSE150 cells.

Chromosomal rearrangements and involved genes have been reported to play important roles in the development and progression of human malignancies. But the gene rearrangements in esophageal squamous cell carcinoma (ESCC) remain to be identified. In the present study, array-based comparative genomic hybridization (array-CGH) was performed on the ESCC cell line KYSE150. Eight disrupted genes were detected according to the obviously distinct unbalanced breakpoints. The splitting of these genes was validated by dual-color fluorescence in-situ hybridization (FISH). By using rapid amplification of cDNA ends (RACE), genome walking and sequencing analysis, we further identified gene disruptions and rearrangements. A fusion transcript DTL-1q42.2 was derived from an intrachromosomal rearrangement of chromosome 1. Highly amplified segments of DTL and PTPRD were self-rearranged. The sequences on either side of the junctions possess micro-homology with each other. FISH results indicated that the split DTL and PTPRD were also involved in comprising parts of the derivative chromosomes resulted from t(1q;9p;12p) and t(9;1;9). Further, we found that regions harboring DTL (1q32.3) and PTPRD (9p23) were also splitting in ESCC tumors. The data supplement significant information on the existing genetic background of KYSE150, which may be used as a model for studying these gene rearrangements.

Overexpression of focal adhesion kinase correlates with increased lymph node metastasis and poor prognosis in non-small-cell lung cancer.

BACKGROUND: The aim of this study was to investigate whether focal adhesion kinase (FAK) overexpression correlates with lymph node metastases and prognosis. METHODS: The protein expression of FAK was investigated in 153 paraffin-embedded tissues by immunohistochemical analysis and then correlated with various clinicopathologic parameters. FAK mRNA level was detected with quantitative RT-PCR in 57 NSCLC frozen tissues and 20 normal matched tissues. RESULTS: Immunohistochemistry showed FAK overexpression was significantly associated with positive lymph node metastasis and more advanced disease stage of NSCLCs and adenocarcinoma subtype; real-time PCR also indicated a statistically significant correlation between increased FAK mRNA level and the presence of nodal metastases. Moreover, in survival analysis, FAK overexpression was significantly associated with worse overall survival. CONCLUSIONS: FAK overexpression is a promising pathological factor to predict aggressive behavior and prognosis in patients with NSCLC, particularly in the adenocarcinoma subtype.

The Raf-1 inhibitor GW5074 and the ERK1/2 pathway inhibitor U0126 ameliorate PC12 cells apoptosis induced by 6-hydroxydopamine.

6-Hydroxydopamine (6-OHDA) is a widely used dopaminergic neurotoxin that leads to cell apoptosis in vivo and in vitro, and is a widely accepted experimental model of neurodegeneration in Parkinson's disease. However, the molecular mechanisms responsible for 6-OHDA-induced cell apoptosis are unclear. We found that the treatment of PC12 cells with 6-OHDA resulted in a significant decrease in cell viability and elevated apoptosis as detected by MTT assay, Hoechst 33258 staining, and flow cytometry. In addition, 6-OHDA induced a time-dependent phosphorylation of ERK1/2 at Thr-202/Tyr-204 and of Raf-1 at Ser-338, but a decreased level of Raf-1 phosphorylation at Ser-259. Phosphorylation of ERK1/2 at Thr-202/Tyr-204 and Raf-1 at Ser-338 were inhibited by the Raf-1 inhibitor GW5074, while the ERK1/2 pathway inhibitor U0126 decreased phosphorylation of ERK1/2. Furthermore, 6-OHDA-induced PC12 cells apoptosis was suppressed by GW5074 and U0126. Our results suggest that GW5074 and U0126 act as neuroprotants against 6-OHDA toxicity in PC12 cells by modulating Raf-1/ERK1/2 signaling systems.

Characterization of genetic rearrangements in esophageal squamous carcinoma cell lines by a combination of M-FISH and array-CGH: further confirmation of some split genomic regions in primary tumors.

BACKGROUND: Chromosomal and genomic aberrations are common features of human cancers. However, chromosomal numerical and structural aberrations, breakpoints and disrupted genes have yet to be identified in esophageal squamous cell carcinoma (ESCC). METHODS: Using multiplex-fluorescence in situ hybridization (M-FISH) and oligo array-based comparative hybridization (array-CGH), we identified aberrations and breakpoints in six ESCC cell lines. Furthermore, we detected recurrent breakpoints in primary tumors by dual-color FISH. RESULTS: M-FISH and array-CGH results revealed complex numerical and structural aberrations. Frequent gains occurred at 3q26.33-qter, 5p14.1-p11, 7pter-p12.3, 8q24.13-q24.21, 9q31.1-qter, 11p13-p11, 11q11-q13.4, 17q23.3-qter, 18pter-p11, 19 and 20q13.32-qter. Losses were frequent at 18q21.1-qter. Breakpoints that clustered within 1 or 2 Mb were identified, including 9p21.3, 11q13.3-q13.4, 15q25.3 and 3q28. By dual-color FISH, we observed that several recurrent breakpoint regions in cell lines were also present in ESCC tumors. In particular, breakpoints clustered at 11q13.3-q13.4 were identified in 43.3% (58/134) of ESCC tumors. Both 11q13.3-q13.4 splitting and amplification were significantly correlated with lymph node metastasis (LNM) (P = 0.004 and 0.022) and advanced stages (P = 0.004 and 0.039). Multivariate logistic regression analysis revealed that only 11q13.3-q13.4 splitting was an independent predictor for LNM (P = 0.026). CONCLUSIONS: The combination of M-FISH and array-CGH helps produce more accurate karyotypes. Our data provide significant, detailed information for appropriate uses of these ESCC cell lines for cytogenetic and molecular biological studies. The aberrations and breakpoints detected in both the cell lines and primary tumors will contribute to identify affected genes involved in the development and progression of ESCC.

Calcium overload induces C6 rat glioma cell apoptosis in sonodynamic therapy.

PURPOSE: Our aim was to study calcium overload-induced apoptosis and its relation to reactive oxygen species (ROS) in rat C6 glioma cells after sonodynamic treatment (SDT). MATERIALS AND METHODS: Hematoporphyrin monomethyl ether (HMME) was used as the sonosensitizer. The concentration of intracellular Ca(2+) ([Ca(2+)](i)) was measured by fluorometry. Apoptosis and necrosis rates were evaluated by a flow cytometry. Moreover, sarcoplasmic reticulum Ca(2+) -ATPase (SERCA(2)), cytochrome c (cyto-c) and cleaved caspase-3 were investigated by immunoblotting. RESULTS: Our study indicated that [Ca(2 +)](i) and ROS increased in cells of SDT group, the apoptosis rate, quantity of cyto-c and cleaved caspase-3 markedly increased after SDT. Furthermore, N-Acetyl-L-cysteine (NAC) or 1,2-bisethane-N,N,N',N'-tetraacetic acid tetrakis ester (BAPTA-AM) could decrease the apoptosis rate, the release of cyto-c and cleaved caspase-3 in SDT group, SERCA(2) degradation was found in SDT group and could also be prevented by the addition of NAC. CONCLUSIONS: Our results show that HMME-SDT can induce C6 cell death through both necrosis and apoptosis. ROS in C6 cells play a decisive role in HMME-SDT-induced cell death. The endoplasmic reticulum (ER) may be a major target of HMME-SDT, ROS can induce SERCA(2) degradation, causing the elevation of [Ca(2+)](i).

Comparison of the antiangiogenic activity of modified RGDRGD-endostatin to endostatin delivered by gene transfer in vivo rabbit neovascularization model.

PURPOSE: Endostatin plays an important role in inhibiting corneal neovascularization (CNV). The aim of this study was to evaluate the antiangiogenic activities of lipid-mediated subconjunctival injection of the modified RGDRGD (arginine- glycin- aspartic- arginine- glycin- aspartic- endostatin gene in a rabbit model of neovascularization in vivo. METHODS: A modified human endostatin gene containing an RGDRGD motif was obtained by rapid site-directed mutagenesis. Forty New Zealand white rabbits underwent alkaline burn and developed CNV, which were randomly divided into four groups: an experimental control group, a PCI empty vector group, a PCI-endostatin group, and a PCI-RGDRGD-endostatin group. The vector, endostatin, and RGDRGD-endostatin groups received injections into the superior bulbar conjunctiva after the burn. An injection of 5 µg was given twice at 1-week intervals. Four eyes of two rabbits received neither treatment nor alkaline burn and served as absolute normal controls. The areas of CNV were monitored after 7 and 14 days. Corneas were examined by histology, and VEGF (vascular endothelial growth factor) and CD31 (platelet endothelial cell adhesion molecule-1) expression was detected by immunohistochemistry after 7 and 14 days. Retina, liver, and kidney were examined by histology, and CD38 expression in the inflammatory cells was detected by immunohistochemistry at 90 days. RESULTS: Subconjunctival injection of both native endostatin and modified RGDRGD-endostatin genes resulted in a significant suppression of CNV in vivo, with modified RGDRGD-endostatin being more effective than native endostatin. The mean concentration of VEGF in the PCI-RGDRGD-endostatin group significantly decreased compared to the means in the other groups. Upon histological examination, the endostatin-treated and RGDRGD-endostatin-treated eyes showed significantly less neovascular area and fewer vessels than the control and vector-injected groups. Retinal, hepatic, and renal tissue sections were normal, and there was no inflammatory cell infiltration observed. CONCLUSIONS: Native and modified endostatin can significantly inhibit CNV by suppressing the expression of VEGF. However, modified endostatin with the RGDRGD motif is far more effective than the endostatin gene in antiangiogenic activity.

Downregulated expression of inhibitor of growth 4 (ING4) in advanced colorectal cancers: a non-randomized experimental study.

Colorectal cancer has a high cure rate if it can be detected early. Identifying and understanding the genes involved may enable early diagnosis and reduce mortality. The aim of our study was to investigate the correlation between the expression of ING4 and the pathological features in patients with colorectal cancer. We assayed ING4 protein expression levels in tumor samples from 97 patients diagnosed with colorectal cancer between January 2001 and January 2002. The patients received no other treatments except surgery. ING4 protein expression was downregulated in adenoma relative to normal mucosa and further reduced in colorectal cancer tissues. Furthermore, the suppression of ING4 expression was also related to the more advanced Dukes' stages. We observed that ING4 expression levels in patients with lymphatic metastasis were lower than those without metastasis. Together, our results indicate that ING4 play a role in colorectal carcinoma progression.

Inhibitory effect of biopolymer materials on scar formation following trabeculectomy.

AIM: To investigate the inhibitory effects of amniotic membrane, polylactic acid membrane and chitosan membrane on scar formation following trabeculectomy. METHODS: A total of 24 New Zealand white rabbits (48 eyes) were randomly divided into 4 groups: amniotic membrane group, polylactic acid membrane group, chitosan membrane group, and control group, with 6 rabbits (12 eyes) in each group. The left eyes underwent routine trabeculectomy, and the right eyes were considered as controls. Amniotic membrane, polylactic acid membrane and chitosan membrane were respectively installed under sclera flap in three groups, but any treatment was not applied in control group. Intraocular pressure, conjunctival filtering bleb, and anterior chamber inflammation responses were monitored at day 1, 3, 7, 14, 28 and 56 post-operatively. Eyeball tissue underwent histopathological examination at day 56 post-operatively. RESULTS: Fibrocytes and inflammatory cells were reduced in amniotic membrane, polylactic acid membrane and chitosan membrane groups compared to that in control group. At day 1 post-operatively, intraocular pressure was decreased in three membrane groups compared to that in control group. At day 14 post-operatively, the intraocular pressure was decreased significantly, while it of three membrane groups was significantly lower than that of preoperative (P<0.01). There were no significant differences among three membrane groups (P>0.05). Filtering bleb of four groups was clearly observed at day 7 post-operatively, but there was no significant difference in pair-wise comparison. At day 28 and 56 post-operatively, filtering bleb in control group was significantly narrowed compared to that in three membrane groups (P<0.05), but there was no significant difference in pair-wise comparison of three membrane groups. CONCLUSION: All amniotic membrane, polylactic acid membrane and chitosan membrane can effectively inhibit scar formation following trabeculectomy, the effect of amniotic membrane is the best.

[Inhibitory effect of transforming growth factor-ß(1) on oral squamous cell carcinoma brain metastasis Tb cell line].

OBJECTIVE: To investigate the inhibitory effect of transforming growth factor (TGF)-ß1 on oral squamous cell carcinoma (OSCC) Tb cell line. METHODS: Cell counting method was used to examine the inhibitory effect of TGF-ß1 on Tb cell and flow cytometry (FCM) assay performed to measure the changes of cell cycle. Superarray was used to screen the changing expression of genes in TGF-ß1/Smads signaling pathway.RT-PCR method was used to detect the results of Superarray. RESULTS: TGF-ß1 showed significant inhibiting effect on OSCC Tb cell line. TGF-ß1 blocked the cell cycle at G1 phase. The expression level of activin receptor-like kinase-1 (ACVRL-1), anti-mullerian hirmine (AMH), cyclim-dependent kinase inhibitor-2B (CDKN-2B) and transforming growth factor-beta-indnced factor (TGIF) was higer in the cells treated with TGF-ß1 than in control, while TDGF-1 expression was down-regulated. ACVRL-1 and CDKN-2B gene expression was consistent with the results of Superarray. CONCLUSIONS: TGF-ß1 can inhibit the growth of OSCC Tb cell line. The mechanism may be related to the regulation of cell cycle and the expression of ACVRL-1 and CDKN-2B in TGF-ß1-Smads signaling pathway.

Down-regulation of ING4 is associated with initiation and progression of lung cancer.

AIMS: Tumour suppressor ING4 is one of ING family genes, which are involved in cell cycle arrest, gene transcription regulation, DNA repair and apoptosis. ING4 inhibition has been reported in various tumours, including gliomas, breast tumours, and stomach adenocarcinoma. The aim was to evaluate ING4 expression in lung cancers. METHOD AND RESULTS: By immunohistochemistry of 246 lung tumour tissues, reduced ING4 nuclear and cytoplasmic expression were both revealed in lung cancer and associated with tumour grade. Interestingly, compared with normal tissues, we found more tumours with ING4 expression in the cytoplasm higher than in the nucleus. Nuclear ING4 inhibition correlated with the tumour stage and lymph node metastasis. Consistent with these findings, semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting demonstrated decreased ING4 mRNA and expression in 100% (50/50) tumour tissues. Furthermore, ING4 expression was lower in grade III than in grades I-II tumours. Reduced ING4 mRNA correlated with lymph node metastasis. CONCLUSIONS: Our results indicate that overall inhibition of ING4 expression and ING4 expression higher in cytoplasm than in nucleus of tumour cells may be involved in the initiation and progression of lung cancers, and thus, analysis for ING4 expression may be useful as a clinical diagnostic and prognostic tool for lung cancer.

RUNX3 inhibits cell proliferation and induces apoptosis by TGF-beta-dependent and -independent mechanisms in human colon carcinoma cells.

BACKGROUND: Genes involved in the TGF-beta signaling pathway are often altered in several types of cancers. The TGF-beta-resistant human colon cancer cell line HT-29 has inactivated TbetaRII and deficient expression of RUNX3 and Smad4, which are involved in the TGF-beta signaling pathway. METHODS: Western blot and immunocytochemistry were performed to confirm gene expression, the MTT assay to detect cell growth, flow cytometry to investigate the cell cycle and the TUNEL to detect cell apoptosis. RESULTS: In the absence of TGF-beta, Bim was upregulated, cell growth was inhibited and apoptosis was induced. TGF-beta treatment did not affect RUNX3 expression; however, the increase in Bim expression was significant and time dependent. Interestingly, Smad4 but not Smad2/3 was also upregulated upon exposure to TGF-beta. This was not the case after TGF-beta treatment of parent HT-29 cells. As expected, TGF-beta further inhibited cell growth and induced apoptosis in HT-29/RUNX3+ cells. CONCLUSION: Our data demonstrate that RUNX3 is involved in TGF-beta-dependent and -independent cell growth inhibition and apoptosis induction pathways.

[Functional significance of TGF-beta1 signal transduction pathway in oral squamous cell carcinoma].

OBJECTIVE: The aim of this study was to investigate the expression of transforming growth factor-beta1 (TGF-beta1) and its signaling pathway molecules in oral squamous cell carcinoma (OSCC) and analyze the association between these factors and genesis and metastasis of OSCC. METHODS: The express of TGF-beta1, TbetaRI, TbetaRII and Smad4, a pivotal downstream molecule of its signaling, in 10 normal oral mucosa tissues and 108 OSCC was detected by SP immunohistochemistry, and thier correlation with genesis and metastasis of OSCC were assessed. RESULTS: The expressions of TbetaRII and Smad4 were lower in the tumors (34.3%, 38.9%) than those in the normal oral epithelium (80.0%, 100.0%, P < 0.05). The positive expression rates of TGF-beta1 and TbetaRI in the normal oral epithelium and OSCC were not significantly different (P > 0.05). There was an inverse correlation between TGF-beta1, Smad4, TbetaRII, TbetaRI expression and clinical stages (P < 0.01). The expression of TGF-beta1 was related with histological differentiation and tumor localization (P < 0.05). There was a relationship beteween Smad4 expression and histological differentiation and lymph node metastasis (P < 0.05). The expression of TbetaRII in the samples with lymph node metastasis was less than that in the ones without lymph node metastasis (P < 0.01), although there was no association between expression of TbetaRII and lymph node metastasis status. CONCLUSION: There is an important relationship between the abnormal TGF-beta1/Smad4 signal pathway and genesis and development of OSCC, while the low expressed Smad4 and TbetaRII may promote the metastasis of OSCC.

Fibroblast growth factor receptor 2-positive fibroblasts provide a suitable microenvironment for tumor development and progression in esophageal carcinoma.

PURPOSE: Tumor fibroblasts (TF) have been suggested to play an essential role in the complex process of tumor-stroma interactions and tumorigenesis. The aim of the present study was to investigate the specific role of TF in the esophageal cancer microenvironment. EXPERIMENTAL DESIGN: An Affymetrix expression microarray was used to compare gene expression profiles between six pairs of TFs and normal fibroblasts from esophageal squamous cell carcinoma (ESCC). Differentially expressed genes were identified, and a subset was evaluated by quantitative real-time PCR and immunohistochemistry. RESULTS: About 43% (126 of 292) of known deregulated genes in TFs were associated with cell proliferation, extracellular matrix remodeling, and immune response. Up-regulation of fibroblast growth factor receptor 2 (FGFR2), which showed the most significant change, was detected in all six tested TFs compared with their paired normal fibroblasts. A further study found that FGFR2-positive fibroblasts were only observed inside the tumor tissues and not in tumor-surrounding stromal tissues, suggesting that FGFR2 could be used as a TF-specific marker in ESCC. Moreover, the conditioned medium from TFs was found to be able to promote ESCC tumor cell growth, migration, and invasion in vitro. CONCLUSIONS: Our study provides new candidate genes for the esophageal cancer microenvironment. Based on our results, we hypothesize that FGFR2(+)-TFs might provide cancer cells with a suitable microenvironment via secretion of proteins that could promote cancer development and progression through stimulation of cancer cell proliferation, induction of angiogenesis, inhibition of cell adhesion, enhancement of cell mobility, and promotion of the epithelial-mesenchymal transition.

Reduced expression and novel splice variants of ING4 in human gastric adenocarcinoma.

ING4, a new member of the ING (inhibitor of growth) family of tumour suppressor genes, has been found to be deleted or down-regulated in gliomas, breast tumours, and head and neck squamous cell carcinomas. The goal of the present study was to investigate whether the expression and alternative splicing of ING4 transcripts are involved in the initiation and progression of stomach adenocarcinoma. ING4 mRNA and protein expression was examined in gastric adenocarcinoma tissues and human gastric adenocarcinoma cell lines by RT-PCR, real-time RT-PCR, tissue microarray immunohistochemistry, and western blot analysis. Alterations in ING4 transcripts were determined through sequence analysis of ING4 cDNA. Our data showed that ING4 mRNA and protein were dramatically reduced in stomach adenocarcinoma cell lines and tissues, and significantly less in female than in male patients. We also found that reduced ING4 mRNA expression correlated with the stage of the tumour. Interestingly, by sequence analysis, we discovered five novel aberrantly spliced variant forms of ING4_v1 and ING4_v2. These variants cause a codon frame-shift and, eventually, deletion of the NLS or PHD domain contributing to the mislocalization of p53 and/or HAT/HDAC complexes and, subsequently, altered gene expression in gastric adenocarcinoma. These results suggest that attenuated and aberrant ING4 expression may be involved in the initiation and progression of stomach adenocarcinoma.

Effects of cloned tumstatin-related and angiogenesis-inhibitory peptides on proliferation and apoptosis of endothelial cells.

BACKGROUND: Tumstatin is a recently developed endogenous vascular endothelial growth inhibitor that can be applied as an anti-angiogenesis and antineoplastic agent. The study aimed to design and synthesize the small molecular angiogenesis inhibition-related peptide (peptide 21), to replicate the structural and functional features of the active zone of angiogenesis inhibition using tumstatin and to prove that synthesized peptide 21 has a similar activity: specifically inhibiting tumor angiogenesis like tumstatin. METHODS: Peptide 21 was designed and synthesized using biological engineering technology. To determine its biological action, the human umbilical vein endothelial cell line ECV304, the human ovarian cancer cell line SKOV-3 and the mouse embryo-derived NIH3T3 fibroblasts were used in in vitro experiments to determine the effect of peptide 21 on proliferation of the three cell lines using the MTT test and growth curves. Transmission electron microscopy (TEM) and flow cytometry (FCM) were applied to analyze the peptide 21-induced apoptosis of the three cell lines qualitatively and quantitatively. In animal experiments, tumor models in nude mice subcutaneously grafted with SKOV-3 were used to observe the effects of peptide 21 on tumor weight, size and microvessel density (MVD). To initially investigate the role of peptide 21, the effect of peptide 21 on the expression of vascular endothelial growth factors (VEGFs) by tumor tissue was semi-quantitatively analyzed. RESULTS: The in vitro MTT test and growth curves all indicated that cloned peptide 21 could specifically inhibit ECV304 proliferation in a dose-dependent manner (P < 0.01); TEM and FCM showed that peptide 21 could specifically induce ECV304 apoptosis (P < 0.01). Results of in vivo experiments showed that tumors in the peptide 21 group grew more slowly. The weight and size of the tumors after 21 days of treatment were smaller than those in the control group (P < 0.05), with a mean tumor inhibition rate of 67.86%; MVD of the tumor tissue in the peptide 21 group was significantly lower than in the control group (P < 0.05); the number of cells positive for VEGF in the peptide 21 group was significantly fewer than in the control group (P < 0.01). CONCLUSIONS: Similar to the activity of tumstatin in specifically inhibiting tumor angiogenesis, peptide 21 may specifically inhibit tumor endothelial cell proliferation and induce their apoptosis, thereby suppressing tumor angiogenesis and indirectly inhibit the growth, infiltration and metastasis of tumors. Peptide 21 may exert its effect through down-regulating the VEGF expression of tumor cells and vascular endothelial cells.