Senolytic cocktail dasatinib plus quercetin enhances the antitumor effect of senescence-inducing radiotherapy in a preclinical model of melanoma

Abstract

Purpose/Objective(s): Although radiation is beneficial in killing tumor cells, it also causes accumulation of senescent cells in tumors and in the surrounding healthy tissue. Senescent tumor cells can recover by acquiring a senescence-associated secretory phenotype (SASP), which leads to undesired tumor cell proliferation. We hypothesize that the clearance of RT-induced senescence by a senolytic cocktail Dasatinib + Quercetin (DQ), may enhance the radio-sensitivity to RT in melanoma. Here, we present data on the effect of DQ and RT on tumor cells and senescence in a radio-resistant melanoma model in vitro and in vivo. Materials/Methods: Exponentially growing melanoma cells (B16F10) were irradiated with 2 Gy, 6 Gy or 12 Gy X-rays at room temperature. Following irradiation, cells were cultured for 2, 3, 5 and 8 days and markers for senescence were determined. In an in vivo study, C57BL/6 mice were injected with 0.5 × 106 B16F10 cells subcutaneously in the right leg. Mice were irradiated with three fractions of 10 Gy, and Dasatinib (5 mg/kg) + Quercetin (50 mg/kg) was given daily by oral gavage beginning from either 1 day after RT (early stage) or 7 days after RT (later stage) for total of 5 doses. Tumor volumes and survival were recorded. Tumor cell senescence was determined by the cells’ morphology. Expression of biomarkers for senescence in tumor tissues were determined by senescence associated β-galactosidase (SA-β-Gal) assay, DNA damage (53BP1/ γ-H2AX foci), and qRT-PCR analysis for p21 and p16 genes, and SASP including IL-1b, IL-6, IL-8, TGF-b, TNF-a, and CXCL10. Results: In vitro, RT increased signals for SA-β-Gal and volume of tumor cells in a dose dependent fashion: 0 Gy (0%) vs. 2 Gy (10%) vs. 6 Gy (50%) vs. 12 Gy (85%). QRT-PCR showed a time and dose-dependent increase of expression for CXCL10 and p21, which was higher at 12 Gy after 8 days compared to other doses and time points (all P < 0.05). In B16F10 tumor bearing mice treatment with RT + DQ (7 days post RT, late stage) reduced tumor volumes and extended survival compared to controls and groups treated with RT or DQ only. In contrast, the combination of RT + DQ on day 1 post RT (early stage) failed to enhance antitumor effects and survival. Analysis of biomarkers for senescence in irradiated tumors showed reduced expression of TGF-b and p21 in RT + DQ (7 days post RT, later stage) vs. RT alone. SA-β-Gal staining was reduced in the 53BP1/ γ-H2AX positive area in the RT + DQ (later stage) group compared to RT alone. Conclusion: The combination of senescence-inducing RT and a senolytic cocktail DQ enhanced the antitumor effects in a mouse model for melanoma in a time-dependent fashion. Further studies are ongoing to assess a potential mechanism of clearance of RT-induced senescence to validate clinical studies.