Multidrug resistance protein 5 (MRP5/ABCC5) is a 161 kDa member of the super family of ATP-binding cassette (ABC) superfamily of transmembrane transporters that is clinically relevant for its ability to confer multidr...Multidrug resistance protein 5 (MRP5/ABCC5) is a 161 kDa member of the super family of ATP-binding cassette (ABC) superfamily of transmembrane transporters that is clinically relevant for its ability to confer multidrug resistance by actively e?uxing anticancer drugs. ABCC5 has also been identified as an efflux transporter of cGMP (cyclic guanosine monophosphate). Elevated intracellular levels of cGMP in cancer cells have been implicated in several clinical studies, that may induce apoptosis, and as a result many different cancer cells seem to overcome this deleterious effect by increased efflux of cGMP through ABCC5. Thus inhibition of ABCC5 may have cytotoxic effects mediated through cGMP and it will also increase the intracellular concentration of other drugs that are aimed for the treatment of cancer which are otherwise exported out of the cells. Considering the functional importance and lack of X-ray crystal structure of ABCC5, present work was undertaken to construct 3D structure of protein using homology modeling protocol of YASARA structure (V. 16.3.28). In this study, five different ABC templates (PDB ID’s: 4F4C, 4Q9H, 4M1M, 4M2T and 4KSD) were used for homology modeling. Five models were constructed on each template and a hybrid model was built using all five templates. All models were refined and ranked as per their overall Z-score. The top ranked ABBC5 model was based on template 4Q9H that had 91.2% of residues in allowed regions as revealed by PROCHECK-NMR and the QMEAN score was 0.54 which indicated a reliable model. The results of the study and the proposed model can be further used for elucidating the structural and functional aspects of ABCC5 and to gain more insights to the molecular basis of ABCC5 inhibition through docking studies.展开更多
[目的]探讨宫颈癌紫杉醇抗性形成的潜在机制。[方法]紫杉醇浓度从0.001μmol/L逐渐递增至0.1μmol/L处理Hela细胞超过12个月进行Hela紫杉醇抗性细胞(Hela/TR)的筛选。检测Hela与Hela/TR细胞的细胞活力、凋亡水平、细胞周期和肿瘤干细胞...[目的]探讨宫颈癌紫杉醇抗性形成的潜在机制。[方法]紫杉醇浓度从0.001μmol/L逐渐递增至0.1μmol/L处理Hela细胞超过12个月进行Hela紫杉醇抗性细胞(Hela/TR)的筛选。检测Hela与Hela/TR细胞的细胞活力、凋亡水平、细胞周期和肿瘤干细胞水平。紫杉醇处理后,检测Hela与Hela/TR细胞内紫杉醇浓度。[结果]紫杉醇处理可以显著抑制Hela细胞的增殖并且诱导细胞凋亡。与Hela细胞相比,Hela/TR细胞中CD44^(+)细胞的比例显著增加,并且Hela/TR细胞中肿瘤干细胞标志物SOX2和ALDH1的表达水平高于Hela细胞。与Hela细胞相比,Hela/TR细胞的细胞内紫杉醇浓度明显降低(0.0759±0.0130 vs 0.0031±0.0004,t=12.52,P<0.05)。与Hela细胞相比,Hela/TR细胞中ABCC5的表达水平显著上升(0.15±0.04 vs 0.72±0.04,t=22.53,P<0.05)。敲低ABCC5后,Hela/TR细胞的细胞内紫杉醇浓度明显升高。与Hela细胞相比,Hela/TR细胞中FOXM1的表达水平上升。敲低FOXM1后,Hela/TR细胞中ABCC5的表达水平下降(0.13±0.07 vs 0.64±0.03,t=14.97,P<0.05),细胞内紫杉醇浓度明显上升。过表达miR-548o-3p后,Hela/TR细胞中FOXM1和ABCC5的水平表达下降,细胞内紫杉醇浓度明显上升(0.003±0.000 vs 0.072±0.010,t=15.43,P<0.05)。miR-548o-3p靶向FOXM1 mRNA的3′端非翻译区。过表达miR-548o-3p后,Hela/TR细胞对紫杉醇的抵抗显著下降(0.51±0.05 vs 0.10±0.01,t=17.98,P<0.05)。[结论]宫颈癌紫杉醇抗性细胞中miR-548o-3p负反馈失调导致FOXM1/ABCC5轴过度激活,提升了紫杉醇从宫颈癌细胞内的排除效率,增强了宫颈癌细胞的活力。展开更多
文摘Multidrug resistance protein 5 (MRP5/ABCC5) is a 161 kDa member of the super family of ATP-binding cassette (ABC) superfamily of transmembrane transporters that is clinically relevant for its ability to confer multidrug resistance by actively e?uxing anticancer drugs. ABCC5 has also been identified as an efflux transporter of cGMP (cyclic guanosine monophosphate). Elevated intracellular levels of cGMP in cancer cells have been implicated in several clinical studies, that may induce apoptosis, and as a result many different cancer cells seem to overcome this deleterious effect by increased efflux of cGMP through ABCC5. Thus inhibition of ABCC5 may have cytotoxic effects mediated through cGMP and it will also increase the intracellular concentration of other drugs that are aimed for the treatment of cancer which are otherwise exported out of the cells. Considering the functional importance and lack of X-ray crystal structure of ABCC5, present work was undertaken to construct 3D structure of protein using homology modeling protocol of YASARA structure (V. 16.3.28). In this study, five different ABC templates (PDB ID’s: 4F4C, 4Q9H, 4M1M, 4M2T and 4KSD) were used for homology modeling. Five models were constructed on each template and a hybrid model was built using all five templates. All models were refined and ranked as per their overall Z-score. The top ranked ABBC5 model was based on template 4Q9H that had 91.2% of residues in allowed regions as revealed by PROCHECK-NMR and the QMEAN score was 0.54 which indicated a reliable model. The results of the study and the proposed model can be further used for elucidating the structural and functional aspects of ABCC5 and to gain more insights to the molecular basis of ABCC5 inhibition through docking studies.
文摘[目的]探讨宫颈癌紫杉醇抗性形成的潜在机制。[方法]紫杉醇浓度从0.001μmol/L逐渐递增至0.1μmol/L处理Hela细胞超过12个月进行Hela紫杉醇抗性细胞(Hela/TR)的筛选。检测Hela与Hela/TR细胞的细胞活力、凋亡水平、细胞周期和肿瘤干细胞水平。紫杉醇处理后,检测Hela与Hela/TR细胞内紫杉醇浓度。[结果]紫杉醇处理可以显著抑制Hela细胞的增殖并且诱导细胞凋亡。与Hela细胞相比,Hela/TR细胞中CD44^(+)细胞的比例显著增加,并且Hela/TR细胞中肿瘤干细胞标志物SOX2和ALDH1的表达水平高于Hela细胞。与Hela细胞相比,Hela/TR细胞的细胞内紫杉醇浓度明显降低(0.0759±0.0130 vs 0.0031±0.0004,t=12.52,P<0.05)。与Hela细胞相比,Hela/TR细胞中ABCC5的表达水平显著上升(0.15±0.04 vs 0.72±0.04,t=22.53,P<0.05)。敲低ABCC5后,Hela/TR细胞的细胞内紫杉醇浓度明显升高。与Hela细胞相比,Hela/TR细胞中FOXM1的表达水平上升。敲低FOXM1后,Hela/TR细胞中ABCC5的表达水平下降(0.13±0.07 vs 0.64±0.03,t=14.97,P<0.05),细胞内紫杉醇浓度明显上升。过表达miR-548o-3p后,Hela/TR细胞中FOXM1和ABCC5的水平表达下降,细胞内紫杉醇浓度明显上升(0.003±0.000 vs 0.072±0.010,t=15.43,P<0.05)。miR-548o-3p靶向FOXM1 mRNA的3′端非翻译区。过表达miR-548o-3p后,Hela/TR细胞对紫杉醇的抵抗显著下降(0.51±0.05 vs 0.10±0.01,t=17.98,P<0.05)。[结论]宫颈癌紫杉醇抗性细胞中miR-548o-3p负反馈失调导致FOXM1/ABCC5轴过度激活,提升了紫杉醇从宫颈癌细胞内的排除效率,增强了宫颈癌细胞的活力。
