Radiosensitization Effect And Mechanisms Of Atovaquone On Nasopharyngeal Carcinoma Cells

[Background and objective]: Nasopharyngeal carcinoma(NPC)is a head and neck carcinoma with a high incidence in southern China and Southeast Asia,but rare in other parts of the world.Xiangxi autonomous prefecture is one of most prevalent areas of NPC in Hunan province.Radiotherapy is the preferred and effective treatment modality for NPC.Radiation resistance is one of important impedient to the improvement of the efficacy of NPC.Currently,there is no evidence of class A-level drug for non-toxicity radiosensitizers in clinical NPC,and some candidate drugs are still in the pre-clinical phase.The sensitization strategy of non-cytotoxic agent is different from chemotherapy,which has the potential to increase the efficacy of radiotherapy.Atovaquone(ATQ)is a broad-spectrum antiprotozoal drug,and recent studies indicates that ATQ inhibits cell growth in multiply cancers through proliferation inhibition,apoptosis induction,and oxidative phosphorylation repression to sensitize chemoradiotherapy in vivo and in vitro,but the effect of ATQ on NPC and the radiosensitising mechanism is largely unclear.[Methods and mate rials]: Multiply fractions of sublethal irradiation were performed by medical linear accelerator to establish radioresistant NPC cell line CNE2-IR and the control CNE2 cell line;the cell viability of NPC cells was determined by thiazole blue colorimetric arrays;the radiosensitivity of NPC cells were detected by radiation clonal survival assay;flow cytometry was used to analyze cell cycle distribution;hoechst33258 staining was used to observe the apoptotic morphology of NPC cells;flow cytometry was used to detect the levels of reactive oxygen species;quantity PCR was used to determine the m RNA expression levels of key signaling molecules of proliferation,apoptosis,DNA demage repair and aerobic glycolysis;western blotting was performed to determine the protein expression level of hub molecules in the proliferation,apoptosis and aerobic glycolysis.[Results]: Radioresistant NPC cell line CNE2-IR and the control CNE2 cell line were screened and confirmed;the areas under the radiaton survival curves of the CNE2-IR and CNE2 were 2.158 and 1.713,respectively,suggesting the successful construction of radioresistant NPC cell lines;thiazole blue colorimetric arrays showed that atovaquone had the similar cytotoxic effects on CNE2 and CNE2-IR in a dose-dependent manner and half inhibition rate(IC50)were 16.4 μmol/L and 17.0 μmol/L,respectively(P< 0.05);the area under the clone survival curve of radiotherapy combined with ATQ showed that a marked increase in the areas under the curves in the CNE2-IR and CNE2 were 0.559 and 0.345,respectively(P< 0.05),sugggesting that 2.5μ mol/L ATQ has a non-cytotoxic radiotherapy sensitization effect on NPC cells in vitro;apoptosis rate by Hoechst33258 dying of radiotherapy combined with ATQ showed a marked increase was visible in CNE2-IR relative to CNE2(P< 0.05),suggesting the low dose of ATQ may specifically induce cell apoptosis in radioresistant NPC cells;flow cytometry showed that a marked increase of G2-M phase in CNE2-IR cells by radiotherapy combined with ATQ compared with the vehicle,suggesting that ATQ increased the re-distribution of cell cycle in radioresistant NPC cells;western blotting analysis showed that a marked decrease in Bcl-2/Bax ratio and phosphorylation of gamma H2 AX,AKT,and ERK levels were observed in CNE2-IR cells by the treatment of radiotherapy combined with ATQ compared to the control(P<0.05),suggesting the low dose of ATQ increase the raditation-induced apoptosis in radioresistant NPC cells;quantity PCR and western blot detection showed that ATQ inhibited the protein levels of HK2 and PKM2 in radioresistant NPC cells;flow cytometry showed that ATQ combined with radiotherapy significantly increased the level of reactive oxygen species in CNE2-IR cel s.[Conclusion]: 1.ATQ exerts a non-cytotoxic radiosensitization activity in NPC in vitro;2.ATQ combined with radiotherapy increases the apoptosis and cycle redistribution in NPC.3.ATQ may inhibit the DNA damage repair,down-regulate the energy supply in cancerous glycolysis pathway,and up-regulate the levels of reactive oxygen species and plays the role as a non-cytotoxic radiosensitization agent in NPC.