Melanoma antigen family A4 (MAGEA4)의 새로운 항암제 내성기전에 관한 연구

Abstract

The melanoma antigen (MAGE) gene family in human consists of 55 members that are classified as type Ⅰ MAGE genes (including MAGEA, MAGEB and MAGEC) and type Ⅱ MAGE genes. Type Ⅰ MAGE genes are highly expressed in various forms of cancer, but not in other normal tissues except for the testes. The doxorubicin-resistant AML-2 cells (AML-2/DX100) have been selected and characterized with respect to drug resistance mechanisms. Interestingly, cDNA microarray, RT-PCR assay and Western blot analysis revealed that AML-2/DX100 cells overexpressed MAGEA4. In this study, to determine whether overexpression of MAGEA4 contributes directly to drug resistance, each MAGEA4 and its mutants were cloned into the mammarian expression vector and then introduced into human embyonic kidney (HEK) 293T and fibrosarcoma HT-1080 cells. MTT assay showed that MAGEA4 transfectants increased resistance to doxorubicin, daunorubicin and hydrogen peroxide, but not to 5-fluorouracil, cisplatin and mitoxantrone. Flow cytometry showed the decreased accumulation of doxorubicin, daunorubicin, rhodamine 123, mitoxantrone and calcein-AM, suggesting that MAGEA4 may function as an efflux pump. Also, doxorubicin accumulation was increased by cyanide-induced ATP depletion in MAGEA4 transfectants. Various P-glycoprotein (Pgp) inhibitors as well as MAGEA4 siRNA in MAGEA4 transfectants and MAGEA4-overexpressing cells increased accumulation of doxorubicin. Immunoprecipitation and Western blot analysis revealed that MAGEA4 neither interacts with Pgp nor alters its expression. Confocal microscopy and subcellular fractionation demonstrated that MAGEA4 was located in plasma membrane, cytosol and nucleus. MAGEA4 protein formed non-disulfide homo- and hetero-dimeric structure. In addition, MAGEA4 had a reactive oxygen species-scavenging activity. These results suggest that acquisition of drug resistance in cancer cells could be at least in part associated with overexpression of MAGEA4 with membrane transporter and antioxidant activities. It is implicated that MAGEAs can be suitable targets for novel therapeutic strategies of MAGEAs-overexpressing cancer cells.