The New England Journal of Medicine

The New England Journal of Medicine. that up-regulated IL-6 plays an important role in the development of EGFR-TKI-induced interstitial fibroblastic proliferation. Therefore, blocking of IL-6 signaling could be beneficial to malignancy patients undergoing EGFR-TKI treatment for reducing the risk of its unfavorable effects. and amplification, and the conversation between EGFR and HER-2 and so on [2-6]. More importantly, EGFR-TKI treatment gives rise to severe side effects, including acute interstitial pneumonia [7]. Although some studies Diethylstilbestrol have suggested risk factors for side effects [8-12], detailed molecular mechanism for their development remains unknown. Recently, Kim indicated that EGFR-TKI activated STAT3 in non-small cell lung malignancy cells [13]. They also showed that STAT3 activation was caused by interleukin-6 (IL-6) in an autocrine manner. IL-6 is one of inflammatory cytokines and is well known as a malignancy progression-related cytokine [14,15]. Because STAT3 is one of the targets for anti-cancer drug resistance [16], most of investigations have been only focused on how IL-6 regulates the drug resistance in EGFR-TKI-treated malignancy cells. In the current study, we explored therapeutic effects of EGFR tyrosine kinase inhibition, using two EGFR-TKIs and an EGFR antibody, in human FASN tongue and lung malignancy cell lines. Further, we found that EGFR blocking could increase IL-6 in the malignancy cells. Because IL-6 has been suggested to contribute to the development or progression of acute interstitial pneumonia [17-20], we anticipated the possible linkage between IL-6 from malignancy cells and EGFR-TKI-induced acute interstitial pneumonia. Our results suggested that IL-6 secreted from EGFR-TKI-treated malignancy cells induced lung fibrosis. Accordingly, a combination of IL-6 pathway blocker and EGFR-TKI may show more favorable effects in malignancy patients. RESULTS EGFR-TKI inhibits the growth of malignancy cell lines We first investigated the growth inhibition effect of EGFR-TKI treatment in human tongue and lung malignancy cells, using MTT assay. AG1478 treatment could decrease the growth of HSC-3 cells dramatically in dose- and time-dependent manners, as compared with mock-treated cells (Physique ?(Figure1A).1A). The growth of A549 cells was similarly inhibited by AG1478 (Physique ?(Figure1B1B). Open in a separate window Physique 1 EGFR-TKI inhibits cell proliferationHSC-3 (A) and A549 (B) cells were treated with different concentrations (1-100 M) of AG1478 for different durations (24-96 hrs). Then, cell proliferation was measured (= 6), using a kit and absorbance at 530 nm or 630 nm. Bars represent common standard deviation (SD) of three impartial experiments. *P 0.001 by student’s test (DMSO-treated cells). To confirm the inhibition of EGF pathway by EGFR-TKI treatment, HSC-3 and A549 cells were treated with EGF after the pre-treatment of AG1478 and EGFR antibody. EGF treatment stimulated EGFR phosphorylation at 10 min (Physique ?(Figure2).2). EGF treatment also increased phosphorylation of STAT3 and Diethylstilbestrol MAPK in HSC-3 cells as well as Akt phosphorylation in A549 cells. When cells were pre-treated with AG1478 or EGFR antibody, EGFR phosphorylation was inhibited especially in HSC-3. AG1478 also inhibited phosphorylation of STAT3, Akt, and MAPK. These results suggest that EGFR-TKI and EGFR antibody decrease cell growth via inhibiting EGF phosphorylation. Open in a separate window Physique 2 EGFR-TKI inhibits phosphorylation of molecules related to downstream signaling of EGFR HSC-3 and A549 cells treated with EGF and other drugs as indicated were analyzed on western blotting, using antibodies to the downstream signaling of EGFR, including pEGFR (175 kDa), EGFR (175 kDa), pSTAT3 (86 kDa), STAT3 (86 kDa), pAKT (60 kDa), AKT (60 kDa), pMAPK (42 and 44 kDa), and MAPK (42 and 44 kDa). -Actin (43 kDa) served as an internal control. Malignancy cells treated with EGFR-TKI secretes IL-6 Western blotting was then performed in HSC-3 cells treated with AG1478 for up to 24 hrs. As shown in Figure ?Physique3A,3A, EGF induced EGFR and STAT3 phosphorylation from 10 min to 6 hrs. Further, AG1478 pre-treatment effectively prevented their phosphorylation induced by EGF activation. On the other hand, AG1478 pre-treatment increased STAT3 phosphorylation at 24 hrs while EGF treatment did not induce the phosphorylation of EGFR and STAT3 at 24 hrs. We also confirmed STAT3 phosphorylation at 24 hrs using another EGFR-TKI, ZD1839 (Physique ?(Figure3B3B). Open in a separate window Physique 3 EGFR-TKI increases Diethylstilbestrol phosphorylation of STAT3 (A) HSC-3 cells treated with EGF and/or AG1478 for up to 24 hrs were analyzed on western blotting, using antibodies to pEGFR (175 kDa), EGFR (175 kDa), pSTAT3 (86 kDa), and STAT3 (86 kDa). (B).