Usefulness of optical coherence tomography to detect central serous chorioretinopathy in monkeys.

Many systemic drugs can induce ocular toxicity and several ocular side-effects have been identified in clinical studies. However, it is difficult to detect ocular toxicity in preclinical studies because of the lack of appropriate evaluation methods. Optical coherence tomography (OCT) is useful because it can provide real-time images throughout a study period, whereas histopathology only provides images of sacrificed animals. Using OCT alongside histopathology, attempts were made to find effective approaches for screening of drug-induced ocular toxicity in monkeys. Such approaches could be used in preclinical studies prior to human trials. Six male cynomolgus monkeys (Macaca fascicularis Raffles) were orally administered one of six candidate MAPK/ERK kinase (MEK) inhibitors. Central serous chorioretinopathy, a known side-effect of such inhibitors, was identified in four monkeys by OCT. Artifacts generated during tissue processing meant that histopathology could not detect edematous changes. Thus, OCT is a useful tool to detect ocular toxicity which cannot be detected by histopathology in preclinical studies.

Dual inhibition of MEK1/2 and EGFR synergistically induces caspase-3-dependent apoptosis in EGFR inhibitor-resistant lung cancer cells via BIM upregulation.

Epidermal growth factor receptor (EGFR) gene mutations activate the KRAS-RAF-MEK-ERK pathway in lung cancer cells. EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib induce apoptosis of cancer cells, but prolonged treatment is often associated with acquired resistance. Here, we identified a novel MEK1/2 inhibitor, CZ0775, and compared its cytotoxic effects to those of AZD6244 (selumetinib) in non-small cell lung cancer (NSCLC) cell lines harboring EGFR mutations. The lapatinib-sensitive HCC827 and PC9 and lapatinib-resistant H1650 and H1975 cell lines showed poor responses to CZ0775 and AZD6244 monotherapy with an IC50 > 10 µM. By contrast, combination treatment with lapatinib and CZ0775 inhibited cell proliferation and produced a 2-fold higher number of annexin V-labeled cells than lapatinib alone in H1975 cells. Furthermore, combination treatment decreased phosphorylated extracellular signal related kinase (p-ERK) and survivin levels and upregulated the expression of the pro-apoptotic protein BIM. siRNA-mediated BIM depletion reduced caspase-3 activity (~40%) in lapatinib and CZ0775 treated H1975 cells. An in vitro ERK activity assay showed that p-ERK levels were approximately a 3-fold lower in H1975 cells treated with CZ0775 and lapatinib combination than in cells treated with lapatinib alone. CZ0775 was more cytotoxic than AZD6244 when used in combination with lapatinib. Our results suggest that combination treatment with CZ0774 and EGFR inhibitors is a promising therapeutic approach for the treatment of EGFR-TKI-resistant lung cancers and its effect is mediated by the inhibition of ERK and the induction of BIM.

Induction of immunoglobulin transcription factor 2 and resistance to MEK inhibitor in melanoma cells.

Primary or acquired resistance to MEK inhibitors has been a barrier to successful treatment with MEK inhibitors in many tumors. In this study, we analyzed genome-wide gene expression profiling data from 6 sensitive and 6 resistant cell lines to identify candidate genes whose expression changes are associated with responses to a MEK inhibitor, selumetinib (AZD6244). Of 62 identified differentially expressed genes, we selected Immunoglobulin Transcription Factor 2, also known as transcription factor 4 as a potential drug resistance marker for further analysis. This was because the ITF-2 expression increase in resistant cell lines was relatively high and a previous study has suggested that ITF-2 functions as an oncogene in human colon cancers. We also established an AZD6244 resistant cell line (M14/AZD-3) from an AZD6244 sensitive M14 cell line. The expression of the ITF-2 was elevated both in primary AZD6244 resistant cell line, LOX-IMVI and acquired resistant cell line, M14/AZD-3. Targeted silencing of ITF-2 by siRNA significantly enhanced susceptibility to AZD6244 in resistant cells. Wnt/ß-catenin pathway was activated through direct interaction of p-ERK and GSK3ß. Our results suggest that up-regulation of the ITF-2 gene expression is associated with cellular resistance to MEK inhibitors, and activation of Wnt signaling pathway through interaction of p-ERK and GSK3ß seems to be a mechanism for increase of ITF-2.