In actual fact, during the H322 cells we failed to observe any improvement

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 In actual fact, during the H322 cells we failed to observe any improvement  Empty In actual fact, during the H322 cells we failed to observe any improvement

Post  jy9202 on Thu Jun 19, 2014 5:44 am

In H322 cell line, the improve in EGFR and HER2 surface expression was dose and time dependent. Western blot analysis of isolated cell surface membrane proteins confirmed the improve of EGFR in erlotinib treated Calu 3 cells. Exploiting the ability of cetuximab and trastuzumab to bind EGFR and HER2, we applied these mAbs as key antibodies [You must be registered and logged in to see this link.] for movement cytometry examination. By this technique, as proven in Figure three, we confirmed the surface density of cetuximab and trastuzumab binding websites, re spectively, on Calu 3, H322 and H292 cells have been greater immediately after one uM erlotinib treatment method. These final results recommend that erlotinib enhanced cell surface expression of EGFR or HER2 on sensitive NSCLC cells, resulting in an increase of mAbs binding to cancer cell surface.

Erlotinib induces EGFR protein stabilization The likelihood that the higher EGFR level observed in Calu 3 cells exposed to erlotinib was because of protein stabilization or elevated synthesis was then explored. As shown in Figure 4A, EGFR degree enhanced soon after 2 h of erlotinib [You must be registered and logged in to see this link.] remedy and reached a plateau after 24 h. On top of that, the utmost level was maintained through time inside the presence on the drug. Even so, following 48 h of erlotinib elimination, EGFR expression was lowered to level comparable to untreated cells. Calu 3 have been also taken care of with erlotinib while in the presence of unique inhibitors of mRNA and protein synthesis.

As proven in Figure 4C, the erlotinib induced EGFR [You must be registered and logged in to see this link.] protein increase was neither influenced by Actynomicin D nor Cycloheximide treat ment indicating that the greater level of EGFR just after erlo tinib therapy can be ascribed to publish transcriptional mechanisms such as protein stabilization. Additionally, we analyzed EGFR transcript level by true time PCR right after erlotinib remedy. Erlotinib did not influence EGFR mRNA degree when when compared to untreated cells. With the aim to clarify why the greater degree of EGFR was induced only in sensitive cells, we then examined the effect of EGFR inhibitors and of inhibitors of MAPK and PI3K AKT mTOR signaling transduction pathways on EGFR accumulation in Calu three cell line. Gefitinib, erlotinib, lapatinib substantially inhibited the phosphorylation of p70S6K and p44 42 and induced a significant maximize in EGFR protein level.

The MEK inhibitor U0126 strongly enhanced EGFR expression, in contrast no enhance while in the EGFR degree was observed after incuba tion with all the inhibitors of PI3K AKT mTOR pathway examined. Effects of erlotinib and cetuximab mixed remedy on NSCLC cell development and antibody dependent cell mediated cytotoxicity We then investigated the result of focusing on EGFR by the two the TKI erlotinib and also the mAb cetuximab in the cell viability assay. We taken care of Calu 3, H322 and H1299 cells with erlotinib, cetuximab or the mixture based upon the schedule erlotinib 24 h followed by the mixture of erlotinib with cetuximab for 72 h. As anticipated Calu three and H322 cells were responsive to erlotinib and cetuximab treatment method, whereas H1299 cells had been resistant to both the single regi mens. Evaluating the experimental mixture points with that anticipated by the Bliss criterion, an additive result was observed only in the Calu three cells.

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