Jiping Qi

Matrine induces programmed cell death and regulates expression of relevant genes based on PCR array analysis in C6 glioma cells

Matrine, one of the main components extracted from Sophora flavescens Ait, has a wide range of pharmacological effects including anti-tumor activities on a number of cancer cell lines. This study has investigated whether matrine could also display anti-tumor action on rat C6 glioma cells. Exposure of C6 cells to matrine resulted in inhibition of proliferation and induction of apoptosis in a dose-dependent manner, as measured by the MTT assay and Flow cytometry. The Annexin V/PI staining further detected the apoptotic cells at both early and late phases of apoptosis. We used AO/EB staining to examine the programmed cell death of matrine-treated C6 cells, and showed that the death rate detected by AO/EB staining was higher than the apoptosis rate measured by Annexin V/PI staining, suggesting that autophagy, the Type II programmed cell death, may be involved in matrine-induced cell death, which was further confirmed by electronic microscopy. To explore the molecular mechanism, an apoptosis real-time PCR array was performed, which has demonstrated that 57 genes were at least 2-fold upregulated, and 11 genes were at least 2-fold downregulated in matrine-treated C6 cells, compared with untreated cells. However, the gene expression profiles could only partly and roughly explain molecular mechanisms of apoptosis and autophagy in matrine-treated C6 cells, thus further investigations are required to confirm the specific molecular pathways and related molecules responsible for the programmed cell death.

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. Copyright

DJ-1 may contribute to metastasis of non-small cell lung cancer

Lung cancer is a leading cause of cancer-related death, about 40% human non-small cell lung cancer (NSCLC) patients showed lymph node involvements. However, the precise mechanism for the metastasis is still not fully understood. This study was to analyze the potential molecular mechanism for lung cancer metastasis. In the current study, proteomics analysis by two-dimensional electrophoresis (2-DE) was performed first to identify the differentially expressed protein between the higher metastasis lung adenocarcinoma cell line Anip973 and the lower metastasis lung adenocarcinoma cell line AGZY83-a. We confirmed the result by RT-PCR, immunoblotting and immunocytochemistry analyses in these two cell lines. Then we examined the expression of the differentially expressed protein in tumor tissues of NSCLC patients by immunoblotting and immunohistochemistry analyses. Using 2-DE analysis, we have identified DJ-1 was expressed higher in the higher metastasis Anip973 compared to the parental cell line AGZY83-a, that was confirmed by RT-PCR, immunoblotting and immunocytochemistry analyses. In NSCLC patients’ tumor tissues study, immunoblotting data showed that, DJ-1 expression level was significantly higher in 72.2% (13/18) of NSCLC tissue samples compared to that in paired normal lung tissues (Pxa0=xa00.044). Immunohistochemistry analysis demonstrated increased DJ-1 expression in 85 NSCLC tumor tissue samples compared with 7 normal lung tissue samples (Pxa0=xa00.044). DJ-1 expression was also found to be significantly correlated with cancer lymphatic metastasis (Pxa0=xa00.039). DJ-1 might contribute to the metastasis of NSCLC.

Growth inhibition induced by transforming growth factor-β1 in human oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a world-wide health problem and its incidence accounts for 1.9–3.5% of all malignant tumors. Transforming growth factor beta/Smads (TGF-β/Smads) signaling pathway plays an important role in oncogenesis, but its function and molecular mechanisms in OSCC remain unclear. Expression of transforming growth factor-β receptor type II (TβRII) and Smad4 was studied by immunohistochemistry in 108 OSCC patients and 10 normal controls. Function and molecular mechanisms of TGF-β/Smads signaling pathway was then investigated in two human tongue squamous carcinoma cell lines with high and low metastasis (Tb and Tca8113) by RT-PCR, Western Blot, immunofluorescence, cell growth curve and flow cytometry (FCM), respectively. TβRII and Smad4 were significantly down-regulated in tumor tissues (with or without lymph node metastasis) compared to normal oral epithelium tissues (Pxa0<xa00.05). TGF-β1 induced arrest of the cell cycle rather than cell death in Tca8113 and Tb cells, and this influence was mediated by the increasing the expression and changing the location of its downstream components of TGF-β1/Smads signaling pathway. TGF-β1 rapidly increased the expression of p15 and p21 in both Tca8113 and Tb cells. TGF-β1 did not increase p27 expression in Tca8113 cells, but p27 expression was increased in Tb cells. These indicated that TGF-β1 induced G1 arrest of cell cycle through a different regulating pathway in Tb cells compared with Tca8113 cells. Thus, we conclude that TGF-β/Smads signaling pathway play a important role on cell growth and metastasis potential in OSCC.

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

BackgroundThe aim of this study was to investigate whether focal adhesion kinase (FAK) overexpression correlates with lymph node metastases and prognosis.MethodsThe 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.ResultsImmunohistochemistry 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.ConclusionsFAK overexpression is a promising pathological factor to predict aggressive behavior and prognosis in patients with NSCLC, particularly in the adenocarcinoma subtype.

Inhibitor of Growth 4 Induces Growth Suppression and Apoptosis in Glioma U87MG

Objective: Inhibitor of growth (ING) 4 is a member of the ING family proteins. It has been shown to play an important role in cell cycle, transcription and oncogenesis, but the molecular mechanism of ING4 on tumor growth inhibition has not yet been elucidated. The goal of this study is to investigate the inhibitory effects of ING4 on gliomas and its mechanism by transduction of ING4 cDNA into glioma U87MG. Methods: The effect and mechanisms of ING4 on proliferation and apoptosis of U87MG were evaluated in vitro by MTT assay, flow-cytometric analysis, TUNEL assay, Western blot analysis and animal experiments. Results: The level of ING4 was markedly reduced in glioma tissues, and the extent of reduction correlated with the progression from lower to higher grades of tumors. It was observed that U87MG with exogenous ING4 gene presented with growth suppression, apoptosis enhancement and the deregulation of cell cycle- or apoptosis-regulating proteins. Conclusion: ING4 has a potential role on the growth suppression and apoptosis enhancement in gliomas U87MG via the activation of mitochondrial-induced apoptotic pathway and the hindrance of the cell cycle progression. The low expression and dysfunction of ING4 might be correlated with the tumorigenesis and progression of gliomas.

Differential expression of PAI-RBP1, C1orf142, and COTL1 in non-small cell lung cancer cell lines with different tumor metastatic potential.

Human non–small cell lung cancer (NSCLC) is one of the most common malignancies in the modern world. Its recurrence is mainly due to its ability to invade and metastasize. However, the precise mechanism for tumor development and metastasis is still not fully understood. To shed light on the development of lung cancer, the human giant cell lung carcinoma cell lines 95D with high metastatic potential and 95C with low metastatic potential were selected in this study. The 2 cell lines originated from the same parental cell and share a similar genetic background. In the current study, we identified 3 differentially expressed proteins in 95C and 95D cell lines, namely, PAI-RBP1, C1orf142, and COTL1, by using 2-dimensional electrophoresis proteomics analysis. We found that PAI-RBP1 and C1orf142 expression levels were higher in 95D than in 95C cells, whereas COTL1 expression level was lower in 95D when compared to 95C cells. We also confirmed these results by reverse transcription–polymerase chain reaction and immunoblotting analyses. The messenger RNA and protein levels of PAI-RBP1 and C1orf142 were much higher in 95D than in 95C cells, and COTL1 expression level was lower in 95D than in 95C cells. The PAI-RBP1 expression was assessed by immunohistochemistry in 70 NSCLC and 7 normal lung tissue samples from patients. PAI-RBP1 expression level was higher in tumor tissues (positive staining in 87.1% of cases [61/70]) than in normal tissues (positive staining in 14.3% of cases [1/7]). In conclusion, by studying protein expression in NSCLC cell lines with high and low metastasis as well as in human lung cancer tissues, we have identified 3 proteins, namely, PAI-RBP1, C1orf142, and COTL1, which were differentially expressed in NSCLC cell lines with different metastatic potential. In addition, we also found that PAI-RBP1 might contribute to NSCLC development.

Identification of Novel Subregions of LOH in Gastric Cancer and Analysis of the HIC1 and TOB1 Tumor Suppressor Genes in These Subregions

Previously, we identified 3 overlapping regions showing loss of heterozygosity (LOH, R1–R3 from 11 to 30 cM) on chromosome 17 in 45 primary gastric cancers (GCs). The data indicated the presence of tumor suppressor genes (TSGs) on chromosome 17 involved in GC. Among the putative TSGs in these regions, HIC1 (in SR1) and TOB1 (in SR3) remain to be examined in GC. By immunohistochemistry (IHC), methylation-specific PCR (MSP) and western blot, we evaluated the expression and regulation status for HIC1 and TOB1 protein in GC. We narrowed down the deletion intervals on chromosome 17 and defined five smaller LOH subregions, SR1–SR5 (0.54 to 3.42 cM), in GC. We found that HIC1 had downregulated expression in 86% (91/106) and was methylated in 87% (26/30) of primary GCs. Of the primary GCs showing downregulation of HIC1 protein, 75% (18/24) had methylated HIC1 gene. TOB1 was either absent or expressed at reduced levels in 75% (73/97) of the GC samples. In addition, a general reduction was found in total and the ratio of unphosphorylated to phosphorylated TOB1 protein levels in the differentiated GC cell lines. Further analysis revealed significant simultaneous downregulation of both HIC1 and TOB1 protein in GC tissue microarray samples (67%, 52/78) and in primary GCs (65%, 11/17). These results indicate that silencing of HIC1 and TOB1 expression is a common occurrence in GC and may contribute to the development and progression of the disease.

Expression of GLP-1 receptor and CD26 in human thyroid C-cells: The association of thyroid C-cell tumorigenesis with incretin-based medicine

Recent reports have demonstrated that long-term and high dosage treatments with incretin-based medicine, such as hormone glucagon-like peptide-1 (GLP-1) may induce thyroid C-cell pathological changes in rodents, rather than in humans. Doubts regarding the tumorigenic potential of GLP-1 analogues in human thyroid C-cells remain. The present study aimed to determine the expression levels of GLP-1 receptor (GLP-1R) and cluster of differentiation 26 (CD26) in the C-cells of thyroid tissues from non-neoplastic, medullary carcinoma and hyperplasia subjects, and to explore the potential clinical significance. The following cases were analyzed: Medullary thyroid carcinoma (n=62, including 59 paraffin-embedded samples and 3 fresh frozen samples), C-cell hyperplasia (n=20, paraffin-embedded samples) and non-neoplastic thyroid tissue samples (n=7, paraffin-embedded samples). GLP-1R and CD26 expression was detected using immunohistochemical staining and western blotting. There were significant differences in the expression levels of the two markers between medullary thyroid carcinoma and C-cell hyperplasia, in addition to between medullary thyroid carcinoma and non-neoplastic thyroid tissue following immunohistochemical staining. Similar significant differences in the expression of GLP-1R and CD26 were detected using western blot analysis in the medullary thyroid carcinoma compared with non-neoplastic thyroid tissue sectioned from the aforementioned fresh frozen samples. There was a significant negative correlation between GLP-1R and CD26 expression. In addition, the present data indicated that GLP-1R expression was associated with the age of the patients with medullary thyroid carcinoma. These results suggested that GLP-1R and CD26 may be closely associated with the development of thyroid C-cell hyperplasia and medullary thyroid carcinoma, and indicated the importance of being aware of the side effects of incretin medicine.

Human Gastric Adenocarcinoma Allelotype on Chromosomes 17 and 18

Allelic losses of multiple chromosome loci in gastric adenocarcinoma suggest that inactivation of tumour suppressor genes in these regions may be important for tumourigenesis. To define deletion intervals and find candidate tumour suppressor genes involved in gastric adenocarcinoma pathogenesis, a genome-wide search for loss of heterozygosity (LOH) was conducted in 45 patients with primary gastric adenocarcinoma. Investigations using 29 microsatellite markers spanning chromosomes 17 and 18 showed allelic deletion in 29 (64%) specimens at one or more loci. Five LOH overlap regions, three newly identified as deletion regions, were defined: RI, D17S831–D17S921 at 17p12-13.3; RII, D17S1868–D17S787 at 17q21.3-22; RIII, D17S785–D17S928 at 17q25.3; RIV, D18S61–D18S1161 at 18q22; and RV, D18S462–D18S70 at 18q22-q23. Eleven (24%) patients with chromosome 17 allelic loss also showed LOH on 18q, with at least one region of overlapping. LOH mapping showed allelic losses were widespread on both chromosomes and suggests the possibility that multiple tumour suppressor genes, including one or more that are unknown, might be inactivated in the aetiology of gastric adenocarcinoma.