Therapeutic Potential of T-oligo and its Mechanism of Action

Abstract

Traditional chemotherapy is the first treatment option for the majority of cancer patients, but due to harsh and toxic side effects, more targeted therapies are needed. T-oligo is an oligonucleotide homologous to the 3’ overhang of the telomere. It induces several DNA damage and anti-cancer responses similar to experimental telomere loop disruption, including senescence, apoptosis and differentiation in malignant cells. To explore T-oligo’s anticancer potential, a panel of 6 malignant melanoma cell lines was treated with T-oligo. Melanoma cell lines with functional p53 or p73 exhibited cell death ranging from 11.80% to 31.73% after T-oligo treatment, with MU and MM-AN melanoma cells expressing the maximum response. There was no significant response in p53 and p73 null RPM-EP cells. Based on these results, MM-AN cells were chosen as a model system to study T-oligo’s effects in vitro and in vivo. To further elucidate its mechanism of action, pro-apoptotic and differentiation markers typically up regulated in responsive melanoma cell lines were studied in RPM-EP cells. FACS analysis and immune fluorescence studies confirmed uptake of fluorescein labeled T-oligo in both cell lines. Western blotting and confocal microscopy studies indicated up regulation of YH2AX after T-oligo treatment in MM-AN cells. In RPM-EP cells, expression of p73 and TRP-1 was not detected, nor was there up regulation of E2F1 and Tyrosinase. For in vivo experiments, SCID mice were injected with MM-AN cells to form tumors on their flanks, which were later treated with T-oligo and complementary oligo using Alzet pumps. Results demonstrated a 98% reduction in tumor size, as well as up regulation of differentiation markers important for anti-tumor immune responses. This study provides novel evidence which further establishes p53/p73 as crucial downstream signalling proteins and important players in T-oligo mediated anti-cancer effects in melanoma. Our results clearly demonstrate that T-oligo may be an effective and novel therapeutic for melanoma.