Core Fucosylation Of Copper Transporter 1 Plays A Crucial Role In Cisplatin Resistance Of Ovarian Cancer By Regulating Drug Uptake

Epithelial ovarian cancer is the most lethal gynecological malignancy worldwide.The standard treatment protocol for the initial management of ovarian cancers is cytoreductive surgery,followed by primary chemotherapy with a platinum-based regimen.Approximately 80% of patients will have a complete clinical response to this initial therapy.Although it has a high response rate,epithelial ovarian cancer has high resistance and poor prognosis.The 5-year survival rate of patients with advanced ovarian cancer is less than 30%.Although reduced cellular accumulation of platinum-based drugs,enhanced detoxification capability,aberrant apoptosis pathways and increased DNA repair ability from DNA damage have been proposed,the precise mechanisms how EOC cells achieve resistance to platinum drugs have not been fully identified.The inhibition of cisplatin(c DDP)uptake is a main reason for the c DDP resistance of EOC,which usually occurs during the malignant transformation in ovarian tumors.Copper transporter 1(CTR1)on the cell membrane is a transporter that plays a key role in regulating copper homeostasis,and is also responsible for the uptake of c DDP in yeast and mammals.CTR1 is the glycoprotein of core fucoidylated fucoidylation.Core fucose glycosylation is carried out under the catalysis of core fucose transferase(Fut 8).Core fucoidylation plays an important physiological role in the cell by changing the conformation,location and expression of glycoprotein.Core fucoylation can regulate the interaction between cell surface protein CTR1 and platinum,thereby affecting the platinum uptake and drug resistance of cancer cells.CTR1-mediated c DDP uptake was depended on its metal-binding ecto-domain,which is linked with the N-glycan.However,the role of core fucosylation of CTR1 in c DDP-uptake and in platinum resistance in ovarian cancer has not been elucidated.Objective: To find the correlation between CTR1 glycosylation level and cisplatin resistance in ovarian cancer cells.Methods: The difference of core fucosylation between serum of ovarian cancer patients and normal persons was detected by lectin blot.In ovarian cancer cells,the difference between cisplatin-sensitive cells and cisplatin-resistant cells was detected by lectin blot,q PCR,MTT,cell cycle and apoptosis.In the pathological sections of ovarian cancer patients,immunohistochemical method was used to detect the difference of CTR1 expression between cancer tissues and paracancerous tissues.At the same time,flow cytometry,immunoprecipitation,inductively coupled plasma mass spectrometry(ICP-MS)and detection of the interaction between CTR1 and cisplatin were used to determine the interaction between core fucosylation of CTR1 and cisplatin.Then,the relationship between core fucose and cisplatin resistance was further verified by constructing specific cell lines and restoring models of core fucose knockout.Finally,phosphorylation experiments were used to detect the relationship between core fucose and cisplatin resistance.Results: High level of core fucosylation frequently occurs in c DDP-treated EOC patients.In ovarian cancer cell lines,the core fucoylation level of cisplatin resistant cells was significantly higher than that of cisplatin sensitive cells,and after cisplatin treatment,the survival rate and apoptosis rate of drug-resistant cells were low,and G2/M phase arrest was not obvious.No significant differences in the expression of CTR1 in two kinds of cells,but after immunoprecipitation of CTR1 core fucosylation level of cisplatin resistant cells than cisplatin sensitive cell.Moreover,the interaction between cisplatin resistant cell CTR1 and cisplatin was significantly lower than that of cisplatin sensitive cells.In cisplatin resistant cells,the absorption of platinum was significantly lower than that of cisplatin sensitive cells.In the mouse ovarian epithelial cells,the absence of core fucose promoted the uptake of cisplatin by cells and weakened the resistance to cisplatin.Conclusion: The core fucosylation of CTR1 was significantly upregulated in c DDP-resistant cells compared to the c DDP-sensitive cells.Intriguingly,the hyper core fucosylation suppressed the CTR1-c DDP interactions and c DDP uptake in c DDP-resistant cells.Conversely,contrast to the Fut8+/+ mouse ovarian epithelial cells,the Fut8-deleted(Fut8-/-)cells obviously showed higher c DDP uptake.Furthermore,the recovered core fucosylation induced the suppression of c DDP uptake in Fut8-restored ovarian epithelial cells.In addition,the core fucosylation regulates the phosphorylation of c DDP resistance-associated molecules,such as AKT,ERK,JNK and m TOR.Our findings suggest that the core fucosylation of CTR1 plays an important role in the cellular c DDP uptake,and thus provide new strategies for improving the c DDP-based chemotherapy of EOC.