ABSTRACT: PURPOSE: To assess the role of Apollon in melanoma resistance to intrinsic and extrinsic pathways of apoptosis and to identify strategies to reduce its expression. EXPERIMENTAL DESIGN: Expression of Apollon was assessed in melanoma cell lines and in surgical specimens.Gene expression profiling, mitochondrial depolarization, caspases activation and apoptosis assays were used to test the effect of Apollon silencing, by siRNA, on melanoma response to different anti-tumor agents. Levels of expression of Apollon and melanoma apoptosis were assessed after tumor treatment with the combination of MEK- and mTOR- inhibitors, or of anti-HLA class II mAbs and anti-tumor agents. RESULTS: Melanoma cells constitutively expressed Apollon, in-vitro and in-vivo. Extent of melanoma apoptosis correlated significantly with Apollon downmodulation upon treatment with a MEK inhibitor, a BRAFV600E-specific inhibitor or the nitrosourea fotemustine. Apollon silencing enhanced melanoma apoptosis in response to the cytotoxic drugs camptothecin, celecoxib, fotemustine and temozolomide, to MEK- and BRAFV600E-specific inhibitors, and to soluble or membrane-bound TRAIL. Mechanistically, Apollon silencing in melanoma cells promoted mitochondrial depolarization, caspase-2, -8, -9 and -3 activation and alteration in the expression of genes related to different cellular functions in response to different anti-tumor agents. Treatment of melanoma cells with combination of MEK and of mTOR inhibitors, or of HLA class II antigen-specific mAb and anti-tumor agents, promoted Apollon downmodulation, associated with enhanced apoptotic responses. CONCLUSIONS: The results suggest that Apollon is a relevant molecule in melanoma resistance to apoptosis, and that strategies aimed at downmodulating Apollon protein may improve melanoma cell death in response to anti-tumor agents that trigger the intrinsic or the extrinsic apoptosis pathways. The human melanoma cell line Me23682 was established in our laboratory from a surgical specimen. Cells were routinely maintained in RPMI medium 1640 (Lonza, Basel, Switzerland) supplemented with 10% FBS (Lonza) and 2 mM glutamine (Lonza). Cells were maintained at 37°C in a water-saturated atmosphere of 5% CO2 in air. 3x106 cells were seeded in 10 cm dishes; after 24 hours, cells were transfected with Apollon-specific siRNA or its unrelated control (Stealth RNAi siRNA, Invitrogen Life Technologies, Camarillo, CA) at a final concentration of 75 nmol/L and then left untreated or treated with fotemustine (Muphoran, Italfarmaco, Milan, Italy) at 150 umol/L for 6 hours, or with the BRAFV600E-specific inhibitor PLX4720 (Selleck Chemicals, Houston, TX) at 500 nmol/L for 8 hours, or with the MEK inhibitor PD0325901 (Cayman Chemicals, Ann Arbor, MI) at 5 nmol/L for 8 hours. Each treatment or combination was performed in triplicate. At the end of treatment, cells were collected and RNA extracted.
