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Abstract
BackgroundThe Mre11-Rad50-Nbs1 (MRN) complex is well known for its crucial role in initiating DNA double strand breaks (DSBs) repair pathways to resistant irradiation (IR) injury and thus facilitating radioresistance which severely reduces radiocurability of nasopharyngeal cancer (NPC). Targeting native cellular MRN function would sensitize NPC cells to IR.MethodsA recombinant adenovirus containing a mutant Rad50 gene (Ad-RAD50) expressing Rad50 zinc hook domain but lacking the ATPase domain and the Mre11 interaction domain was constructed to disrupt native cellular MRN functions. The effects of Ad-RAD50 on the MRN functions were assessed in NPC cells lines using western blot, co-immunoprecipitation and confocal microscopy analyses. The increased radiosensitivity of transient Ad-RAD50 to IR was examined in NPC cells, including MTT assay, colony formation. The molecular mechanisms of radiosensitization were confirmed by neutral comet assay and western bolts. Nude mice subcutaneous injection, tumor growth curve and TUNEL assay were used to evaluate tumor regression and apoptosis in vivo.ResultsRad50 is remarkably upregulated in NPC cells after IR, implying the critical role of Rad50 in MRN functions. The transient expression of this mutant Rad50 decreased the levels of native cellular Rad50, Mre11 and Nbs1, weakened the interactions among these proteins, abrogated the G2/M arrest induced by DSBs and reduced the DNA repair ability in NPC cells. A combination of IR and mutant RAD50 therapy produced significant tumor cytotoxicity in vitro, with a corresponding increase in DNA damage, prevented proliferation and cell viability. Furthermore, Ad-RAD50 sensitized NPC cells to IR by causing dramatic tumor regression and inducing apoptosis in vivo.ConclusionOur findings define a novel therapeutic approach to NPC radiosensitization via targeted native cellular Rad50 disruption.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2190-8) contains supplementary material, which is available to authorized users.
Highlights
The Mre11-Rad50-Nbs1 (MRN) complex is well known for its crucial role in initiating DNA double strand breaks (DSBs) repair pathways to resistant irradiation (IR) injury and facilitating radioresistance which severely reduces radiocurability of nasopharyngeal cancer (NPC)
Wild-type Rad50 is upregulated in NPC cells following IR To avoid the influence of mutations and elucidate the contributions of IR induced DNA-damage, we confirmed the expression of wild-type MRN complex proteins in CNE1 and 5–8 F cells after IR
We found that Rad50 was upregulated in irradiated NPC cells, which confirmed that Rad50 was a crucial factor of MRN complex function in NPC cells
Summary
The Mre11-Rad50-Nbs (MRN) complex is well known for its crucial role in initiating DNA double strand breaks (DSBs) repair pathways to resistant irradiation (IR) injury and facilitating radioresistance which severely reduces radiocurability of nasopharyngeal cancer (NPC). Targeting native cellular MRN function would sensitize NPC cells to IR. Representing only approximately 0.7 % of the global cancer burden [1], nasopharyngeal cancer (NPC) is the leading cancer in the South East Asia and Southern China (30.94/100,000 in males and 13.00/100,000 in females) [2], in Guangdong province, Hong Kong, Philippines and Thailand [1]. Improving the radiotherapy dose alone would affect the anatomic proximity of crucial structures and reduce the life quantity [6]. NPC patients who exhibit radioresistance are a key population that contributes to the observed poor outcomes. Improving the sensitization of NPC to radiotherapy represents a useful solution.
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