Effect of Cymbopogon martini (Roxb.) Will.Watson essential oil on antioxidant activity, immune and intestinal barrier-related function, and gut microbiota in pigeons infected by Candida albicans.

Essential oils are potential alternatives to antibiotics for preventing Candida albicans (C. albicans) infection which is responsible for economic losses in the pigeon industry. Cymbopogon martini essential oil (EO) can inhibit pathogens, particularly fungal pathogens but its potential beneficial effects on C. albicans-infected pigeons remain unclear. Therefore, we investigated the impact of C. martini EO on antioxidant activity, immune response, intestinal barrier function, and intestinal microbiota in C. albicans-infected pigeons. The pigeons were divided into four groups as follows: (1) NC group: C. albicans uninfected/C. martini EO untreated group; (2) PC group: C. albicans infected/C. martini EO untreated group; (3) LPA group: C. albicans infected/1% C. martini EO treated group; and (4) HPA group: C. albicans infected/2% C. martini EO treated group. The pigeons were infected with C. albicans from day of age 35 to 41 and treated with C. martini EO from day of age 42 to 44, with samples collected on day of age 45 for analysis. The results demonstrated that C. martini EO prevented the reduction in the antioxidant enzymes SOD and GSH-Px causes by C. albicans challenge in pigeons. Furthermore, C. martini EO could decrease the relative expression of IL-1ß, TGF-ß, and IL-8 in the ileum, as well as IL-1ß and IL-8 in the crop, while increasing the relative expression of Claudin-1 in the ileum and the crop and Occludin in the ileum in infected pigeons. Although the gut microbiota composition was not significantly affected by C. martini EO, 2% C. martini EO increased the abundance of Alistipes and Pedobacter. In conclusion, the application of 2% C. martini EO not only enhanced the level of antioxidant activity and the expression of genes related to intestinal barrier function but also inhibited inflammatory genes in C. albicans-infected pigeons and increased the abundance of gut bacteria that are resistant to C. albicans.

Downregulation of tumstatin expression by overexpression of ornithine decarboxylase.

Tumor angiogenesis, a pivotal process for cancer growth and metastasis, requires both upregulation of pro­angiogenic molecules and downregulation of anti­angiogenic molecules. Anti-angiogenesis therapy represents a promising way for cancer treatment. Tumstatin, a novel endogenous angiogenesis inhibitor, inhibits endothelial cell proliferation, pathological angiogenesis and tumor growth. Ornithine decarboxylase (ODC), overexpressed in various cancers, is associated with cell transformation, tumor invasion and angiogenesis. We found that the expression of tumstatin was suppressed in ODC-overexpressing human cancer cells and renal carcinoma tissues. We presumed that ODC overexpression may downregulate the expression of tumstatin. To be able to test this hypothesis, we generated HEK293 cells that overexpress ODC (ODC transfectants) and characterized the following experimental groups: PBS-treated group, mock transfectants, ODC transfectants, ODC transfectants transfected with pcDNA-ODCr (an antisense ODC-expressing plasmid) group and putrescine-treated group. The effect of ODC overexpression on tumstatin expression was examined by reverse transcriptase-polymerase chain reaction (RT-PCR), western blot analysis and dual luciferase reporter assay. ODC-overexpressing cells and putrescine-treated cells showed suppressed tumstatin mRNA and protein expression, and decreased tumstatin gene promoter activity. Thus, ODC overexpression suppresses the expression of tumstatin, which may provide fundamental evidence for the combination of anti-angiogenic therapy and conventional therapy for cancer treatment.

Combination of fluvastatin and losartan relieves atherosclerosis and macrophage infiltration in atherosclerotic plaques in rabbits.

AIM: To investigate whether the combination of fluvastatin and losartan synergistically relieve atherosclerosis and plaque inflammation induced by a high-cholesterol diet in rabbits. METHODS: Atherosclerosis was induced with a high-cholesterol diet for 3 months in 36 New Zealand white rabbits. The animals were randomly divided into model group, fluvastatin (10 mg·kg(-1)·d(-1)) group, losartan (25 mg·kg(-1)·d(-1)) group, and fluvastatin plus losartan group. After the 16-week treatments, the blood samples the animals were collected, and the thoracic aortas were examined immunohistochemically. The mRNA and protein expression levels of monocyte chemotactic protein-1 (MCP-1) were measured using RT-PCR and Western blot. RESULTS: Compared to the treatment with losartan or fluvastatin alone, the combined treatment did not produce higher efficacy in reduction of blood cholesterol level. However, the combination did synergistically decrease the intimal and media thickness of thoracic aortas with significantly reduced macrophage infiltration and MCP-1 expression in the plaques. CONCLUSION: The combined treatment with losartan and fluvastatin significantly inhibited atherosclerotic progress and reduced inflammation associated with atherosclerotic plaques.

Downregulation of ornithine decarboxylase by pcDNA-ODCr inhibits gastric cancer cell growth in vitro.

Ornithine decarboxylase (ODC), the first rate-limiting enzyme of polyamine biosynthesis, was found to be associated with cell growth, proliferation and transformation. ODC gene expression in gastric cancer was increased and its level was positively correlated with the degree of malignity of gastric mucosa and development of gastric lesions. In order to evaluate the therapeutic effects of antisense RNA of ODC on gastric cancer, an antisense RNA of ODC expressing plasmid pcDNA-ODCr which delivered a 120 bp fragment complementary to the initiation codon of ODC gene was constructed and transfected to gastric cancer cells SGC7901 and MGC803. Expression of ODC in gastric cancer cells was determined by western blot. Cell proliferation was assessed by MTS assay. Cell cycle was analyzed by flow cytometry and Matrigel assay was performed to assess the ability of gastric cancer cell invasiveness. The results showed that the ODC gene expression in gastric cancer cells transfected with the pcDNA-ODCr was downregulated efficiently. Tumor cell proliferation was suppressed significantly, and cell cycle was arrested at G1 phase. Gastric cancer cells had reduced invasiveness after gene transfer. Our study suggested that antisense RNA of ODC expressing plasmid pcDNA-ODCr had antitumor activity by inhibiting the expression of ODC, and downregulation of ODC expression using a gene therapy approach might be a novel therapeutic strategy for gastric cancer.

Effects of eukaryotic expression plasmid encoding human tumstatin gene on endothelial cells in vitro.

BACKGROUND: Tumstatin is a novel endogenous angiogenesis inhibitor which is widely studied using purified protein. The current study evaluates the antiangiogenic effects of tumstatin-overexpression plasmid in vitro, reveals the mechanism underlying the vascular endothelial cell growth inhibition and searches for a novel method administering tumstatin persistently. METHODS: The eukaryotic expression plasmid pcDNA-tumstatin encoding tumstatin gene was constructed and transfected to human umbilical vein endothelial cell ECV304 and human renal carcinoma cell ACHN. Expression of tumstatin in the two cell lines was determined by RT-PCR and Western blotting. Vascular endothelial cell proliferation was assessed by CCK-8 assay and cell cycle was analyzed by flow cytometry. To investigate the mechanism by which pcDNA-tumstatin inhibited vascular endothelial cell proliferation in vitro, cyclin D1 protein was detected by Western blotting. RESULTS: DNA sequence confirmed that pcDNA-tumstatin was successfully constructed. RT-PCR and Western blotting indicated that tumstatin could express in the two cell lines effectively. After tumstatin gene transfer, ECV304 cell growth was significantly inhibited and the cell cycle was arrested in G1 phase. And Western blotting showed that pcDNA-tumstatin decreased the level of cyclin D1 protein. CONCLUSIONS: Overexpression of tumstatin mediated by pcDNA 3.1 (+) specially inhibited vascular endothelial cells by arresting vascular endothelial cell in G1 phase resulting from downregulation of cyclin D1 and administration of tumstatin using a gene therapy might be a novel strategy for cancer therapy.

RNA interference-mediated silencing of UBCH10 gene inhibits colorectal cancer cell growth in vitro and in vivo.

1. UbcH10 is the cancer-related E2 ubiquitin-conjugating enzyme, and its overexpression has been demonstrated in a variety of malignancies. The aim of the present study is to silence UbcH10 gene by RNA interference (RNAi) and to observe its inhibitory effect on the colorectal cancer cell growth in vitro and in vivo. 2. We constructed the expression vector pGPU6/GFP/Neo/UbcH10-RNAi (pUbcH10-RNAi), which contained a UbcH10 short hairpin RNA expression cassette. Then the UbcH10 gene silencing cell lines LoVo/UbcH10-RNAi and HT-29/UbcH10-RNAi were established. Reverse transcription-polymerase chain reaction and western blot analysis were used to evaluate the expression of the UbcH10 gene. Cell Counting Kit-8 was used to assess properties of tumour cell growth in vitro. Flow cytometry was used to detect the effect of pUbcH10-RNAi on the cell cycle of colorectal cancer cells. Furthermore, the anti-tumour effects of pUbcH10-RNAi were evaluated in vivo in a nude mouse xenografts model. 3. Results demonstrated that UbcH10 gene expression was significantly decreased in pUbcH10-RNAi treated cells. Colorectal cancer cells growth was markedly suppressed in the pUbcH10-RNAi group compared with control conditions and colorectal cancer cells were arrested in the G2-M phase. In vivo, the downregulation of UbcH10 gene expression by pUbcH10-RNAi also inhibited tumour growth in a nude mice xenograft model. 4. Our study suggests that RNA interference-mediated silencing of UbcH10 gene has anti-tumour activity on colorectal cancer and might have therapeutic potential for the treatment of colorectal cancer.

Targeted antitumor effect induced by hTERT promoter mediated ODC antisense adenovirus.

The expression of Ornithine decarboxylase (ODC) which is the first key enzyme of polyamine biosynthesis is increased in cancer cells. We had blocked the polyamine synthesis pathway using the adenoviral-mediated antisense ODC in some cancer cells such as prostate cancers and colorectal cancers. These researches demonstrated that ODC antisense expression could inhibit tumor cell growth. In order to reach the goal of applying the targeting gene therapy in clinical practice, we cloned the antisense ODC RNA which was driven by cancer specific promoter (hTERT promoter; telomerase reverse transcriptase promoter) into the adenovirus vector (rAd-CMV-GFP-hTERTp-ODC). Human cancer cell lines (HepG2, Bel-7402, A549) and normal cell lines (HELF, LO2) were infected separately with rAd-CMV-GFP-hTERTp-ODC as well as with control vector (rAd-CMV-GFP). Luciferase activity assay was performed to determine hTERT promoter activity. Cell growth curves analysis, western blot analysis, flow cytometry analysis and Matrigel invasion assays were performed to assess properties of cell growth and invasiveness. The results showed that there was significant inhibition of ODC expression and cell proliferation in cancer cells treated with rAd-CMV-GFP-hTERTp-ODC compared with cells treated with PBS or rAd-CMV-GFP, and no significant inhibition was detected in normal cells. Our research offers a powerful and safe new therapeutic strategy for cancer targeted treatment.

The expression of tumstatin is down-regulated in renal carcinoma.

Tumstatin is the 28 kDa NC1 domain of the alpha3 chain of type IV collagen that inhibits pathological angiogenesis and suppresses endothelial cell proliferation and tumor growth. In the present paper, we expressed and purified recombinant human tumstatin protein and then prepared the anti-tumstatin polyclonal antibody. To investigate the expression of tumstatin in renal carcinoma, tumstatin protein was detected by western blotting using the prepared anti-tumstatin antibody and tumstatin mRNA levels were assayed by RT-PCR. The results showed that the expression of tumstatin gene was down-regulated in renal carcinoma tissues and cells. Our study suggests that as a novel endogenous angiogenesis inhibitor, tumstatin gene expression may be a marker for diagnosis, therapy and prognosis of renal carcinoma.

Adenovirus vector-mediated upregulation of spermidine /spermine N1-acetyltransferase impairs human gastric cancer growth in vitro and in vivo.

Spermidine/spermine N(1)-acetyltransferase (SSAT) is the rate-limiting step in polyamine catabolism. In a previous study, we constructed a recombinant adenovirus, Ad-SSAT, which can express human SSAT. In the present study, we investigated the effect of upregulated and downregulated SSAT on gastric cancer cells. We found that upregulated SSAT could inhibit the growth of MGC803 and SGC7901 cells, whereas adverse results were found with downregulated SSAT. We further analyzed cell cycle profiles and the expression levels of the major cell cycle regulatory proteins of S phase. The results showed that the growth inhibition was caused by S phase arrest. Ad-SSAT suppressed the expression of cyclin A and nuclear factor E2F1 in MGC803 and SGC7901 cells. We observed the E2F promoter activity caused by Ad-SSAT using a reporter gene assay. We also investigated the antitumorigenicity of upregulated SSAT by Ad-SSAT using a SGC7901 xenograft model in nude mice. Our results suggest that the upregulation of SSAT by Ad-SSAT infection inhibited the growth of gastric cancer in vitro and in vivo. Ad-SSAT arrested gastric cancer cells in S phase, which was mediated through downregulation of the cyclin A-E2F signaling pathway.

[Ornithine decarboxylase and S-adenosylmethionine decarboxylase bi-antisense virus inhibit growth and invasion of esophageal cancer cell line Eca109].

BACKGROUND & OBJECTIVE: Esophageal carcinoma is one of the most common malignant tumors in China. It is reported that the content and biosynthesis of polyamine in esophageal cancer is markedly increased. This study was to investigate inhibitory effects of both antisense ornithine decarboxylase (ODCas) and S-adenosylmethionine bi-antisense (AdoMetDCas) on polyamine biosynthesis, cell proliferation and invasion of esophageal cancer cell line Eca109. METHODS: An adenoviral vector Ad-ODC-AdoMetDCas containing antisense sequences of ODCse and AdoMetDCas was used. Cell proliferation was observed by counting viable cells or BrdU labeling. Protein expressions of ODC and AdoMetDC and the polyamine content in Eca109 cells were measured by Western blot and high performance liquid chromatography (HPLC), respectively. Invasion of Eca109 cells in vitro was detected using Matrigel invasion assay. Furthermore, the anti-proliferation effect of Ad-ODC-AdoMetDCas on Eca109 cell xenografts in nude mice was evaluated. RESULTS: Ad-ODC-AdoMetDCas significantly inhibited proliferation of Eca109 cells(P<0.05) and protein expressions of ODC and AdoMetDC. Transfection of Ad-ODC-AdoMetDCas significantly decreased synthesis of three polyamines in Eca109 cells, which were putrescine (Put), spermidine (Spd) and spermine (Spm) (P<0.05). Ad-ODC-AdoMetDCas dramatically suppressed invasiveness of Eca109 cells in vitro(P<0.05). In addition, compared with Ad-GFP, Ad-ODC-AdoMetDCas significantly suppressed the growth of Eca109 cell xenografts in nude mice. CONCLUSION: Ad-ODC-AdoMetDCas significantly inhibits cell proliferation and invasion in esophageal cancer Eca109 cells.