Construction and preservation of a stable and highly expressed recombinant Helicobacter pylori vacuolating cytotoxin A with apoptotic activity.

BACKGROUND: H. pylori is closely related to the occurrence and development of various digestive gastritis, peptic ulcer and mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori is also a class I carcinogen of gastric cancer. VacA is the only exocrine toxin of H. pylori, which plays a very important role in the pathogenesis of H. pylori. The production of VacA in natural circumstances is complex with heavy workload and low yield. Therefore, it is very important to obtain recombinant VacA protein which is stable and biologically active. This study therefore aims to explore the expression, purification and stable storage of VacA toxin of H. pylori in E.coli, and to provide experimental basis for further exploration of the role of VacA in H. pylori -induced inflammation of cancer. RESULTS: A 2502-bp fragment and VacA gene were identified. An 89.7-kDa VacA34-854 recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay. CONCLUSIONS: A VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9).

lncRNA UCA1 induced by SP1 and SP3 forms a positive feedback loop to facilitate malignant phenotypes of colorectal cancer via targeting miR-495.

AIMS: Long noncoding RNA (LncRNA) urothelial cancer associated 1 (UCA1) was dysregulated in colorectal cancers (CRC) and promoted tumor progression of CRC. The aims of this study are to further investigate the underlying mechanism. MAIN METHODS: Short hairpin RNAs (shRNAs) were applied for gene knockdown. microRNA mimic and pcDNA-UCA1 plasmids were transfected for miR-495 and UCA1 overexpression, respectively. MTT was applied to determine cell viability and sensitivity of 5-fluorouracil (FU). Transwell assays were performed to evaluate cell migration/invasion. Angiogenesis was evaluated by tube formation. Western blotting and quantitative PCR were utilized for protein and mRNA detection, respectively. The interaction of UCA1, miR-495 and SP1/SP3 were explored by dual-luciferase assay. RNA pulldown was adopted to determine the UCA1/miR-495 interaction. KEY FINDINGS: UCA1 was significantly upregulated in CRC tissues. UCA1 enhanced cell proliferation, migration/invasion, angiogenesis, epithelial-mesenchymal transition, and resistance to 5-FU in CRC cell lines. MiR-495 was inversely correlated to the expression of UCA1. The results indicated that UCA1 sponged miR-495, leading to the disinhibition of SP1/SP3 expression. SP1/SP3 induced the expression of DNA methyltransferases and, in turn, contributed to UCA1 mediated tumor-promoting actions. Reduction of SP1/SP3 exerted anti-cancer effects, which can be reversed by forced expression of UCA1. SIGNIFICANCE: UCA1-miR-495-SP1/SP3 axis is dysregulated in CRC and contributed to malignant phenotypes of CRC. UCA1-SP1/SP3 may form a positive feedback loop in CRC.

Periostin induces chemoresistance in colon cancer cells through activation of the PI3K/Akt/survivin pathway.

In the present study, we aimed to explore the effects of periostin, a cell adhesion protein, on chemoresistance in colon cancer cells. Reverse-transcription polymerase chain reaction and Western blot analyses were employed to detect periostin expression in SW480 and HT-29 colon cancer cells treated with oxaliplatin or fluorouracil (5-FU). Small interfering RNA was used to downregulate endogenous periostin. Annexin-V/propidium iodide staining was performed to analyze the effects of periostin on drug-induced apoptosis. The results showed that treatment with oxaliplatin or 5-FU elevated both the mRNA and protein levels of periostin in SW480 and HT-29 cells. Silencing of periostin significantly (P < 0.01) augmented drug-induced apoptosis in colon cancer cells, coupled with enhanced cleavage of caspase-3 and poly(ADP-ribose) polymerase. Mechanistic studies revealed that periostin silencing significantly (P < 0.01) suppressed the expression of survivin, an antiapoptotic protein in colon cancer cells. Enforced expression of survivin repressed drug-induced apoptosis in periostin-depleted SW480 and HT-29 cells. Additionally, periostin overexpression increased the expression of survivin and the phosphorylation of Akt, which was reversed by pretreatment with the phosphatidylinositol 3-kinase (PI3K)-specific inhibitor LY294002. Taken together, our data demonstrate that periostin induces chemoresistance in colon cancer cells through activation of the PI3K/Akt/survivin pathway.

Effects of ethanol extract of Radix Sophorae Flavescentis on activity of colon cancer HT29 cells.

This paper mainly studied the inhibitory effect of total ethanol extract of Radix Sophorae Flavescentis on proliferation of colon cancer HT29 cells. By reflux extraction method and with ethanol as extraction solvent, different extracts were obtained at different ethanol concentrations, different solid-liquid ratios, and at different times. And their inhibitory activities against HT29 cells were compared using MTT assay. The experimental results showed that the extraction processes under three conditions can all draw relatively high inhibition rates. The optimum ethanol extraction process conditions were as follows: a solid-liquid ratio of 1:9, 80 min of heat reflux extraction with 95% ethanol.

A study on the inhibitory effect of Solanum lyratum Thunb extract on Lewis lung carcinoma lines.

The objective of this paper was to observe the effects of Solanum lyratum Thunb extract on tumour inhibition, immune function and survival time of tumour-bearing mice. Lung carcinoma-bearing mouse model was established, the tumour-bearing mice were divided into model group, CTX group, Solanum lyratum Thunb extract high-dose group and low-dose group. By the examination of tumour inhibition rate of Solanum lyratum Thunb extract in Lewis lung carcinoma-bearing mice and determination of the number of NK cells and T cell subsets, the survival rate of tumour-bearing mice was observed. Solanum lyratum Thunb extract had some anti-tumour effect in Lewis tumour-bearing mice. The tumour inhibition rate of high-dose group reached 46.28%, and the tumour inhibition rate of low-dose group was 31.42%. Solanum lyratum Thunb extract can improve the NK cell activity of Lewis tumour-bearing mice, increase the number of CD4 cells in the tumour-bearing mice, and significantly increase the survival rate of tumour-bearing mice. The study concluded that Solanum lyratum Thunb extract has some anti-tumour effect and can improve immune function and survival rate of tumour-bearing mice.

[Intrasplenic tumor model of nude mice in the anti- metastasis roles of NGX6 gene against colon cancer].

OBJECTIVE: To establish a liver metastasis model of nude mice in colon cancer so as to determine the function of NGX6. METHODS: The cells of Group HT-29, pcDNA3.1(+)/HT-29, and pcDNA3.1(+)/NGX6/HT-29 were implanted into the spleen of nude mice, respectively. Everyday we measured the weight of the nude mice and observed their ingestion, movement and mental status. The nude mice were killed after 45 days, and the effect of NGX6 on the malignant behavior of HT-29 was assessed by this experiment. RESULTS: In contrast to the other two groups, the metastasis in the liver and xenograft tumor in the spleen of pcDNA3.1(+)/NGX6/HT-29 group was significantly reduced (P<0.01). CONCLUSION: The metastasis of HT-29 colon cancer cell line was significantly inhibited by NGX6 gene. This model of liver metastasis in the nude mice is a proper model to determine the anti-metastasis mechanism of NGX6 gene.

[Effect of NGX6 on gene expression profile of colon carcinoma cell line HT-29].

BACKGROUND & OBJECTIVE: Colon cancer is one of the most common malignant tumors, but the mechanism involved in its pathogenesis is not fully understood. Under the foundation of early study, we intended to explore the effects of NGX6 on the gene expression profile of colon carcinoma cell line HT-29. METHODS: pcDNA3.1-NGX6-transfected HT-29 cells were used as the test, and empty vector-transfected HT-29 cells were used as the control. Differentially expressed genes were screened with high-throughout BiostarH-80sx1 DNA microarray; part of the results of DNA microarray was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: NGX6 transfection induced wide changes of the gene expression profile of HT-29 cells. A total of 377 genes were up-regulated or down-regulated by more than 3 folds, which were involved in cell signal transduction, cell cycle controlling, cell apoptosis, cytokinetics, gene transcription and translation, DNA damage and repairing, protein synthesis, metabolism, and so on. Some of them belonged to some important signal pathways related with cell proliferation and metastasis, such as DDK1, ILK, MMP-1 and COL11A1 in Wnt/beta-catenin signal pathway, ILK in ILK signal pathway, EpHB2 in Eph-Ephrins signal pathway, ROCK2 in RhoA signal pathway, RANBP1 in RanGTPase signal pathway, and RBBP1 in RB/RBBP1 signal pathway. CONCLUSION: NGX6 transfection leads to molecular changes of some important signal pathways, and may suppress cell proliferation and metastasis of tumor by regulating these signal pathways.