ABSTRACT
Objective To investigate the role of ATP￣binding cassette ( ABC ) family on the resistance of nasopharyngeal carcinoma (NPC) stem cells (CSCs) to cisplatin. Methods We compared the differences between the drug extravasation capability of CNE￣2 and CNE￣2S by using Rhodamine￣123 efflux assay. We determined the mRNA and protein expression levels of ABC transport family members, including ABCA3,ABCB1,ABCB5,ABCC1,ABCC2 and ABCG2,after 48 h being treated with 1 μmol.L-1 cisplatin by RT￣PC and Western blotting.Rhoamine￣123 efflux and apoptosis by cisplatin in two kinds of cells was examined by ABCA3 gene silencing with specific small￣interfering RNA. Results The IC50 of cisplatin on CNE￣2S was 4.1 fold to that on CNE￣2(P<0.05).For the relative drug effluent activity and Na+K+ ATPase activity,CNE￣2S was 4.8 fold to CNE￣2(P<0.05),suggested that CNE￣2S expressed more ABCA3,ABCB1,ABCC1 and ABCG2 in comparison to CNE￣2(P<0.05).After 48 h treatment with 1 μmol.L-1 cisplatin,ABCA3 specifically highly expressed in CNE￣2S (P<0.05), and knocking down of ABCA3 resulted in reduction of rhodamine￣123 efflux and increase of apoptosis. Conclusion The cisplatin resistance of NPC CSCs is associated with enhanced expression of ABCA3,ABCC1 and ABCG2, suppression of ABCA3 could reverse the resistance of NPC CSCs to cisplatin.
ABSTRACT
Objective To investigate the way that nasopharyngeal carcinoma (NPC) and NPC stem cells develops resistance to cisplatin through anti-reactive oxygen species mechanism. Methods Using CCK-8 cell counting kit, we measured the half inhibitory concentration of cisplatin against NPC cellsCNE-2and NPC stem cellsCNE-2S, and compared their resistant index. We examined the differences in the reactive oxygen species (ROS) levels, total glutathi?one (GSH) levels, and total superoxide dismutase (SOD) levels between CNE-2 and CNE-2S at different concentrations of cisplatin administration (0.1,0.5 and 1.0μmol·L-1). Using q-PCR, we determined the mRNA expression level of GSS, GCLC, GCLM, SOD1 and SOD2 after 48 hours administration of cisplatin at 1 μmol · L-1. Protein expression level of SOD2 was also tested using Western Blot after 48 hours administration of cisplatin at 1μmol · L-1. Upon silencing the SOD2 in NPC cell through siRNA, Trypan blue was used to analyze cell survival after cisplatin was administrated at 1μmol · L-1. Results The inhibition concentration of cisplatin against CNE-2 was higher than that against CNE-2S (μmol · L-1:9.8 ± 1.1 vs 2.4 ± 0.6,P<0.05). ROS levels in CNE-2 and CNE-2S both rise with cisplatin administration, but ROS levels of CNE-2 before and after cisplatin treatment were both higher than those in CNE-2S (P<0.05). The total gluta?thione levels in CNE-2 and CNE-2S were both increased after 1μmol·L-1 cisplatin treatment but there is no significant dif?ference in levels of glutathione between these two cell lines. After treated with cisplatin, SOD level were increased in both CNE-2S and CNE-2, but it is higher in CNE-2S than that in CNE-2 (P<0.05). The mRNA levels of GSS, GCLC, GCLM, and SOD1 were not different significantly between in CNE-2 and in CNE-2S with or without cisplatin treatment. However, SOD2 in CNE-2S were higher than that in CNE-2 on both mRNA and protein levels (P<0.05). Silenced SOD2 disrupted the resistance of cisplatin in CNE-2S. Conclusion These data suggest that NPC stem cells (CNE-2S) enhance its drug re?sistance to cisplatin through highly expression of SOD2 which posed anti-ROS capacity.