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Home » Archive » 2015 » Veterinary Session

Veterinary session

In vitro pharmacological characterization of selective TMPRSS2 inhibitor
Czimmermann Ágnes Eszter - year 4
SzIU, Faculty of Veterinary Science, Department of Pharmacology and Toxicology
Supervisor: Erzsebet Pasztine Gere PhD

Abstract:

TMPRSS2 is a membrane-bound serine protease enzyme, which plays role in influenza viral infections and in metastatic processes in case of certain tumours. The aim of our study is to characterize the interaction between synthetic 3-amidinophenylalanine TMPRSS2 inhibitor, I-432 and intestinal epithelium in addition to the determination of in vitro efficiency in suppression of metastatic tumour growth.

During our research work the mode of action of I-432 was investigated in non- tumorigenic, porcine jejunal cell monolayer, IPEC-J2 cells cultured on membrane insert. Cytotoxic effect of I-432 was determined by Neutral Red method. The expression of TMPRSS2 was detected by immunofluorescent staining and Western blot. The protease activity of TMPRSS2 was quantified using fluorogenic substrate by fluorimeter. The analysis of hydrogen peroxide concentration was performed by Amplex red assay in IPEC-J2.

Transmigration experiments including the application of breast cancer cell line, MDA-MB-231 coupled with hCMEC/D3 human brain endothelial cells were accomplished to evaluate changes in transmigration tendency.

Neutral red uptake assay proved that cell viability did not change in cells treated with I-432 inhibitor compared to that of control groups. The loss of the expression of active serine protease domain of TMPRSS2 in I-432 treated IPEC-J2 cell was detected. It was also observed that that apical application of I-432 at 10-50 µM concentrations caused reduction in enzymatic activity of TMPRSS2. After incubation of the IPEC-J2 cells with I-432 for 48 hours we detected partial loss of TMPRSS2 immunofluorescent signal due to the altered distribution pattern. Hydrogen- peroxide level analyzed with Amplex red assay did not indicate oxidative stress during and after application of selective TMPRSS2 inhibitor. It was also ascertained that the I-432 did not have any significant impact on the migration of the breast cancer cells through the brain endothelial cells.

Based on our results, I-432 inhibited protease activity of TMPRSS2 successfully in the supernatants of the IPEC-J2 cells. It was proven firstly by us that the expression of active serine protease of TMPRSS2 was significantly reduced in IPEC-J2 cells. It was established that I-432 did not lead to suppression of transmigration processes of breast cancer cells through brain endothelial cells. Our findings characterized pharmacological effects of TMPRSS2 blockage in IPEC-J2 cells which can facilitate the elucidation of I-432 properties in other studies in connection with certain tumour types and influenza viral infections.



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