n-Butylidenephthalide induced apoptosis in the A549 human lung adenocarcinoma cell line by coupled down-regulation of AP-2alpha and telomerase activity.

AIM: To investigate the role of hTERT gene expression and AP-2alpha in n-butylidenephthalide (n-BP)-induced apoptosis in A549 lung cancer cells. METHODS: Viability of A549 cells was measured by MTT assay. Protein expression was determined by Western blot. Telomerase activity was measured using the modified telomere repeat amplification protocol (TRAP) assay. Xenograft mice were used as a model system to study the cytotoxic effect of n-BP in vivo. The morphology of tumor was examined by immunohistochemical staining. RESULTS: The growth of A549 lung cancer cells treated with n-BP was significantly inhibited. Telomerase activity and hTERT mRNA expression were determined by telomeric repeat amplification protocol and reverse transcription-polymerase chain reaction, respectively. n-BP inhibited telomerase activity and hTERT mRNA expression in A549 cells while overexpression of hTERT could abolish BP-induced growth inhibition in the A549 cells. We also showed that hTERT promoter activity in the presence of n-BP was mediated via AP-2alpha. We saw an inhibition of tumor growth when nude mice carrying A549 subcutaneous xenograft tumors were treated with n-BP. Immunohistochemistry of this tumor tissue also showed a decrease in the expression of hTERT. CONCLUSION: The antiproliferative effects of n-BP on A549 cells in vitro and in vivo suggest a novel clinical application of this compound in the treatment of lung cancers.

Chronic NMDA administration to rats up-regulates frontal cortex cytosolic phospholipase A2 and its transcription factor, activator protein-2.

Excessive N-methyl-D-aspartate (NMDA) signaling is thought to contribute to bipolar disorder symptoms. Lithium and carbamazepine, effective against bipolar mania, are reported in rats to reduce brain transcription of an arachidonic acid selective calcium-dependent cytosolic phospholipase A(2) (cPLA(2)), as well as expression of one of its transcription factors, activator protein (AP)-2. In this study, we determined if chronic administration of NMDA (25 mg/kg i.p.) to rats would increase brain cPLA(2) and AP-2 expression, as these antimanic drugs are known to down-regulate excessive NMDA signaling. Administration of a daily subconvulsive dose of NMDA to rats for 21 days decreased frontal cortex NMDA receptor (NR)-1 and NR-3A subunits and increased cPLA(2) activity, phosphorylation, protein, and mRNA levels. The activity and protein levels of secretory phospholipase A(2) or calcium-independent phospholipase A(2) were not changed significantly. Chronic NMDA also increased the DNA-binding activity of AP-2 and the protein levels of its alpha and beta subunits. These changes were absent following acute (3 h earlier) NMDA administration. The changes, opposite to those found following chronic lithium or carbamazepine, are consistent with up-regulated arachidonic acid release due to excessive NR signaling and may be a contributing factor to bipolar mania.

Adenoviral mediated interferon-alpha 2b gene therapy suppresses the pro-angiogenic effect of vascular endothelial growth factor in superficial bladder cancer.

PURPOSE: Intravesical adenovirus mediated interferon-alpha gene transfer has a potent therapeutic effect against superficial human bladder carcinoma xenografts growing in the bladder of athymic nude mice. We determined whether the inhibition of angiogenesis might contribute to the antitumor effect. MATERIALS AND METHODS: We treated several human urothelial carcinoma cells with adenovirus mediated interferon-alpha 2b and monitored its effects on the production of angiogenic factors using real-time reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemical analysis and a gel shift based transcription factor array. To assess the role of adenovirus mediated interferon 2b in angiogenic activity we used in vitro invasion assays and evaluated the anti-angiogenic effects of adenovirus mediated interferon gene therapy in an orthotopic murine model of human superficial bladder cancer. RESULTS: In adenovirus mediated interferon-alpha infected 253J B-V cells vascular endothelial growth factor was decreased and anti-angiogenic interferon-gamma inducible protein 10 was up-regulated. In contrast, the addition of as much as 100,000 IU recombinant interferon had no apparent effect on vascular endothelial growth factor production. Conditioned medium derived from adenovirus mediated interferon 2b infected 253J B-V cells greatly decreased the invasive potential of human endothelial cells and down-regulated their matrix metalloproteinase 2 expression compared to controls. Furthermore, adenovirus mediated interferon 2b blocked pro-angiogenic nuclear signals, such as the transcription factors activating protein-1 and 2, stimulating protein-1, nuclear factor kappaB and c-myb. In vivo experiments revealed significant vascular endothelial growth factor down-regulation and decreased tumor vessel density in the adenovirus mediated interferon 2b treated group compared to controls. CONCLUSIONS: Treatment with adenovirus mediated interferon 2b increases the angiostatic activity of the bladder cancer microenvironment. This inhibition may prove beneficial for treating superficial bladder cancer with adenovirus mediated interferon-alpha and hopefully contribute to a decreased recurrence rate of this neoplasm.