Rosmarinic acid influences collagen, MMPs, TIMPs, glycosylation and MUC1 in CRL-1739 gastric cancer cell line.

Rosmarinic acid (RA) is a natural phenylpropanoid with numerous pharmacological activities. Because of limited studies of the effects of RA action in gastric cancer cells we examined how 100 and 200 µM acid influences MMPs, TIMPs, collagen, MUC1 and specific sugar antigens in gastric adenocarcinoma CRL-1739 cells. We revealed inhibitory effect of RA on MMP-9 activity what was correlated with increased collagen type I expression, main ECM substrate degraded by MMPs. Tissue inhibitor of MMPs, TIMP-1 but not TIMP-2 was significantly decreased on the protein level and increased on mRNA level by RA action what can suggest TIMP-1 independent inhibitory action of an acid on MMP-9 activity. Glycosylation of gastric cancer proteins was also effected by RA. ELISA tests revealed inhibitory effect of an acid on Tn antigen in cell lysates and culture supernatant and on T antigen in cell lysates. RA inhibited also sialylated Tn antigen in protein of culture supernatant and sialyl T in cell lysates. Extracellular domain of MUC1 mucin, main carrier of Tn and T antigens was significantly inhibited by higher dose of RA. The data suggest potential usefulness of RA as a complementary agent supporting chemotherapy in cancer treatment.

Rapid DNA extraction protocol for detection of alpha-1 antitrypsin deficiency from dried blood spots by real-time PCR.

The dried blood spot (DBS) specimens have been successfully employed for the large-scale diagnostics of α1-antitrypsin (AAT) deficiency as an easy to collect and transport alternative to plasma/serum. In the present study we propose a fast, efficient, and cost effective protocol of DNA extraction from dried blood spot (DBS) samples that provides sufficient quantity and quality of DNA and effectively eliminates any natural PCR inhibitors, allowing for successful AAT genotyping by real-time PCR and direct sequencing. DNA extracted from 84 DBS samples from chronic obstructive pulmonary disease patients was genotyped for AAT deficiency variants by real-time PCR. The results of DBS AAT genotyping were validated by serum IEF phenotyping and AAT concentration measurement. The proposed protocol allowed successful DNA extraction from all analyzed DBS samples. Both quantity and quality of DNA were sufficient for further real-time PCR and, if necessary, for genetic sequence analysis. A 100% concordance between AAT DBS genotypes and serum phenotypes in positive detection of two major deficiency S- and Z- alleles was achieved. Both assays, DBS AAT genotyping by real-time PCR and serum AAT phenotyping by IEF, positively identified PI*S and PI*Z allele in 8 out of the 84 (9.5%) and 16 out of 84 (19.0%) patients, respectively. In conclusion, the proposed protocol noticeably reduces the costs and the hand-on-time of DBS samples preparation providing genomic DNA of sufficient quantity and quality for further real-time PCR or genetic sequence analysis. Consequently, it is ideally suited for large-scale AAT deficiency screening programs and should be method of choice.

Cytotoxicity evaluation of three light-cured dentin adhesive materials on human gingival fibroblasts, ex vivo.

PURPOSE: To evaluate the cytotoxic effects of three current light-cured dentin adhesives, in both uncured and post-cured conditions, on human gingival fibroblasts. MATERIAL AND METHODS: The materials tested were Heliobond, Adper Single Bond 2 and Xeno V, which are characterized by various compositions and application procedures. Each agent, in volumes of 5 and 10 µL, was tested after polymerization, and those unpolymerized were diluted in DMEM to 10-3 and 10-5. The cytotoxicity of the adhesives was assessed on the basis of a test of cell viability in a culture of human gingival fibroblasts, with the use of tetrazolic salt (MTT assay). RESULTS: The results showed that, among the adhesive/bonding systems tested, Xeno V was the least cytotoxic. There were statistically significant differences in cell survival between polymerized Xeno V, Adper Single Bond 2 and Heliobond in the amount of 5 µL as well as between the Xeno V and Adper Single Bond 2 in 10-5 dilutions. The tested adhesives were more toxic in the polymerized form than in the dilutions. Samples of 10 µL resulted in a lower survival percentage of fibroblasts compared to 5 µL. CONCLUSION: All the tested adhesives demonstrated cytopathic effects towards human gingival fibroblasts, but varied in their cytotoxicity. This has clinical implications. Dentists should follow the rules of adhesive application, precisely dose them and not allow direct contact with the gums as, even after polymerization, adhesive agents exhibit potential cytotoxic activity.