Assessment of plasma and tissue fibronectin EIIIB splice variant expressions measured serially using RT-PCR in a wound model of rabbits.

BACKGROUND: Fibronectin (FN) is an indispensable part of the extracellular matrix. During regeneration or wound healing, the plasma form of FN is incorporated into the fibrin clots to form a temporary fibrin-FN matrix, and also locally synthesized cellular FN migrates to the clot to regenerate the injured tissue. We aimed to examine wound tissue FN EIIIB and plasma FN EIIIB expression levels in an experimental wound healing model in rabbits. METHODS: Plasma and tissue EIIIB splice variant expressions were measured serially with RT-qPCR in a cutaneous wound model of rabbits. RESULTS: Tissue FN expression increased as beginning on day 3 and continued to increase on days 6 and 9, reaching maximum expression at day 12 before starting to decrease. On the contrary to the tissue levels, plasma FN levels gradually decreased until day 15 when expression returned to the initial values. CONCLUSION: The findings of the current study support that tissue EIIIB expression level increases during wound healing; and plasma EIIIB expression level decreases minimal changed. This is in contrast to reports where plasma FN provisionally helps ECM formation. Therefore, our data show an essential role of EIIIB at the tissue level in accelerating the wound healing process. The RT-qPCR method in our experimental setup can provide more accurate and precise results compared to the antibody-based methods.

Design and synthesis of pyrimidinyl acyl thioureas as novel Hsp90 inhibitors in invasive ductal breast cancer and its bone metastasis.

Invasive ductal carcinoma is the most common breast malignancies tumors and has tendency to bone metastases. Many oncogenic client proteins involved in formation of metastatic pathways. Stabilization, regulation, and maintenance of these oncogenic client proteins are provided with Heat Shock Protein 90 (Hsp90). Hsp90 perform these processes through its ATP binding and subsequent hydrolysis energy. Therefore, designing Hsp90 inhibitors is a novel cancer treatment method. However, many Hsp90 inhibitors have solubility problems and showed adverse effects in clinical trials. Thus, we designed and synthesized novel pyrimidinyl acyl thiourea derivatives to selectively inhibit Hsp90 alpha in human invasive ductal breast (MCF-7) and human bone osteosarcoma (Saos-2) cell lines. In vitro experiments showed that the compounds inhibited cell proliferation, ATP hydrolysis, and exhibited cytotoxic effect on these cancer cell lines. Further, gene expression was analyzed by microarray studies on MCF-7 cell lines. Several genes that play vital roles in breast cancer pathogenesis displayed altered gene expression in the presence of a selected pyrimidinyl acyl thiourea compound. Molecular docking studies were also performed to determine interaction between Hsp90 ATPase domain and pyrimidinyl acyl thiourea derivatives. The results indicated that the compounds are able to interact with Hsp90 ATP binding pocket and inhibit ATPase function. The designed compounds powerfully inhibit Hsp90 by an average of 1 µM inhibition constant. And further, the compounds perturb Hsp90 N terminal domain proper orientation and ATP may not provide required conformational change for Hsp90 function as evidenced by in silico experiments. Therefore, the designed compounds effectively inhibited both invasive ductal breast carcinoma and bone metastasis. Pyrimidinyl acyl thiourea derivatives may provide a drug template for effective treatment of invasive ductal breast carcinoma and its bone metastasis as well as new therapeutic perspective for drug design.