変異型EGFR発現細胞株への活性化EGFR下流シグナル導入によるgefitinib耐性化

Patients with pulmonary adenocarcinoma carrying the epidermal growth factor receptor (EGFR) mutation tend to display dramatic clinical response to treatment with the EGFR tyrosine kinase inhibitor gefitinib. Unfortunately, in many cases the cancer cells eventually acquire resistance, and this limits the duration of efficacy. To gain insight into these acquired resistance mechanisms, we first prepared HEK293T cell line stably transfected with either wild-type (WT) or mutant (L858R) EGFR, and then expressed oncogenic K-Ras12V mutant in the latter transfectant. Although 293T cells expressing wild-type EGFR did not show any growth inhibition by gefitinib treatment similarly to the non-transfected cells, the cells expressing the EGFR-L858R were exquisitely sensitive. Consistently, phospho-Akt levels were decreased in response to gefitinib in cells expressing EGFR-L858R but not in cells with EGFR-WT. In contrast, 293T cells expressing both EGFR-L858R and oncogenic K-Ras were able to proliferate even in the presence of high concentration of gefitinib probably by inducing Erk1/2 activation. We also expressed K-Ras12V in the gefitinib-sensitive pulmonary adenocarcinoma cell line PC-9, which harbors an in-frame deletion in the EGFR gene. The activated K-Ras inhibited the effects of gefitinib treatment on cell growth, cell death induction and levels of phospho-Akt, as well as phospho-Erk. These data indicate that activated Ras could substitute most of the upstream EGFR signal, and are consistent with the hypothesis that mutational activation of targets immediately downstream from the EGFR could induce the secondary resistance to gefitinib in patients with lung cancer carrying EGFR mutation

INHIBITION OF IFN-γ PRODUCTION BY SOLUBLE FACTORS DERIVED FROM ORAL CANCER CELLS

The streptococcal antitumor agent OK-432 is commonly used as an immunopotentiator for immunotherapy in various types of malignant tumors including oral cancer. It has been demonstrated that OK-432 elicits an antitumor effect by stimulating immunocompetent cells, thereby inducing multiple cytokines including interferon (IFN)-γ, interleukin (IL)-2 and IL-12. Serum concentrations of IFN-γ in patients with oral cancer were examined 24 h after administration of OK-432. Serum concentrations of IFN-γ in patients with advanced cancer were significantly lower than those in patients with early cancer. These results suggested that some soluble factors produced by cancer cells may inhibit IFN-γ production with OK-432. Thus, in the present study, an in vitro simulation model was established for the immune status of patients with oral cancer by adding conditioned medium (CM) derived from oral cancer cell lines into a culture of peripheral blood mononuclear cells (PBMCs) derived from a healthy volunteer. We investigated whether soluble factors derived from oral cancer cells affected IFN-γ production from PBMCs following stimulation with OK-432. PBMCs stimulated with OK-432 produced a large amount of IFN-γ; however, both IFN-γ production and cytotoxic activity from PBMCs induced by OK-432 were inhibited by the addition of CM in a dose-dependent manner. In order to examine these inhibitory effects against IFN-γ production, the contribution of inhibitory cytokines such as IL-4, IL-6, IL-10, transforming growth factor-β and vascular endothelial growth factor was investigated. However, neutralization of these inhibitory cytokines did not recover IFN-γ production inhibited by CM. These results indicated that unknown molecules may inhibit IFN-γ production from PBMCs following stimulation with OK-432

Japanese Initiative for Education in Pharmaceutical Medicine and Clinical Research Training

Development of new medicines has become increasingly difficult with less possibility of success in seeds-finding and ever rising operational costs. Failure to comply with ethical standards for human research protection also erodes social trust in clinical development. In order to develop competence of professionals in medicines development such as clinical investigators and drug development scientists, a variety of educational courses and training programs have been developed and executed worldwide. As Japan is no exception and shares the same concerns, significant governmental and non-governmental efforts have been made to invest in the development of academic educational courses and adherence to international standards. This article introduces examples of the adoption of technologies to realize a user-friendly and sustainable learning management as well as an adaptation of syllabuses and core curricula to meet international standards in the era of global medicines development

Zinc transport via ZNT5-6 and ZNT7 is critical for cell surface glycosylphosphatidylinositol-anchored protein expression

Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs [ZNT5-ZNT6 heterodimers (ZNT5-6) and ZNT7-ZNT7 homodimers (ZNT7)], which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway

The Effect of Gefitinib on B-RAF Mutant Non-small Cell Lung Cancer and Transfectants

Abstract:We previously reported one patient with squamous cell carcinoma of the lung that showed the long-term effect to gefitinib with complete response. In the present report, we examine the epidermal growth factor receptor (EGFR) and K-RAS, HER2, and B-RAF mutations in this patient to find a B-RAF exon11 mutation, resulting in a substitution of valine by phenylalanine at codon 470 (V470F) as a novel type of B-RAF mutation in human cancers. In addition, the fluorescence in situ hybridization analysis for EGFR showed the high polysomy status. B-RAF is a nonreceptor serine/threonine kinase whose kinase domain has a structure similar to other protein kinases, including EGFR members. Of interest, the B-RAF V470F mutation corresponds to a position similar to the EGFR G719X mutation located on the phosphate binding (P)-loop of EGFR that clamps ATP into the catalytic cleft. This observation suggests that gefitinib may have an anti-cancer effect on B-RAF mutant tumors. Indeed, previous reports demonstrated that H1666 cells harboring B-RAF G465V mutations showed sensitivity to gefitinib, inhibiting phosphorylation of ERK1/2. We examined the effect of gefitinib on transient transfectants of the B-RAF mutant, but no drastic inhibition of ERK1/2 phosphorylation that was one of the downstream molecules of B-RAF was induced by gefitinib.In summary, we found a novel B-RAF V470F mutation in lung squamous cell carcinoma that showed response to gefitinib. However, our in vitro investigation did not explain the response observed in this particular patient. Further investigation is necessary to elucidate the mechanism of tumor sensitivity to EGFR tyrosine kinase inhibitors