Nattokinase resistance to gefitinib has been associated to PTEN loss, RAS mutations or the upregulation of theinsulin like growth factor 1 receptor or EGFR mutations. In our study, the continuous exposure of PC3 cells to gefitinib resulted in a sustained growth inhibition for about 2 months before the surviving cells resumed proliferation. A stable gefitinib resistant subline was established after four more months. During drug desensitization experiments we observed a time dependent increment of gefitinib IC50. Moreover, gefitinib was still able to induce the cells which had already been treated for 8 weeks, developing resistance undergoing a G0/G1 cell cycle arrest with a corresponding reduction in the G2/M cells without evident cell apoptosis. PC3 cells showed a substantial growth inhibition when initially challenged glycyrrhetin with gefitinib. The surviving population, however, eventually resumed proliferation even though a successful blockade of EGFR signal transduction was evident in these cells.
After the onset of custom peptide synthesis gefitinib resistance, we observed an increased MEK MAPK activity. Inhibition with PD 98059 was able to reduce cell proliferation and induce cell apoptosis in TKI R cells. Chronic treatment with gefitinib was also able to induce strong changes in cell shape, with an increment in the fibroblast like cell proportion, and an increased basal p Her2 expression. No specific Her2 ligands are evident if EGF, TGF and HB EGF bind to the heterodimers of EGFR/Her2 or if heregulins bind to the heterodimers of Her2 containing erbB3 and erbB4. PC3 cells are negative for the Her4 protein but express erbB3. Our observations are in agreement with those of Jain et al, who used an in vivo system to generate TKI R cells from the CWR22R PCa xenograft in which the serial passages into gefitinib treated nude mice produced a gefitinib resistant tumor within three generations. When treated with 2C4 pertuzumab, a humanized monoclonal antibody targeted to an epitope of the Her 2 extracellular domain, TKI R CWR22R tumor growth was inhibited by 60%. The TKI R PC3 PCa cells showed a substantial growth promotion following the challenge with NGF and were MG-341 clearly more responsive to this ligand as compared to the parental cells. In addition, a significant increase in basal TrkA phosphorylation was demonstrated in the gefitinib resistant population compared with the parental cells.
Furthermore, the enhanced role postulated for TrkA in mediating the growth of TKI R PCa was reinforced by dose response studies, in which significant differences in the sensitivity phase to TrkA TKI CEP701 were shown in the TKI R cell line compared with the parental line. Clearly, the prostate gefitinib resistant phenotype compensated for the EGFR blockade via Her2 and/or TrkA signalling. The PC3/TKI R prostate cells increased TrkA expression and NGF sensitivity to TrkA inhibition, whereas the parental PC3 cells were only partially affected by the TrkA inhibitor. On the acquisition of resistance to the EGFR inhibitor, however, PC3 cells acquired 50% growth inhibition in response to the inhibition of TrkA. This implies that there is no modulation or interplay between these pathways in the prostate cells but merely a switch in pathways with the intervention described. In addition, it has been shown that in breast.