Study Of Drug Resistance Through P62Regulating RIP1Ubiquitination In Human Ovarian Cancer Cell

Backgroud:Drug resistance of ovarian cancer cells is a key issue in the treatment of ovariancancer. Previous study has found that the expression of p62protein is increased incisplatin-resistant ovarian cancer cells, which induces autophagy. Autophagydecreased cisplatin sensitivity through relieve endoplasmic stress，resulting incisplatin resistance of ovarian cancer cells. So the imbalance of the survival and thedeath of tumor cells is the main cause of drug resistance.Other studies found that has the ubiquitin-related domain structrully, and canbind with K48polyubiquitin and K63polyubiquitin. Thus p62can not only helpubiquitinated proteins degradation through autophagy, but also act with otherscaffold proteins through polyubiquitin to take part in two-side roles of the survivaland the death. RIP1in the structure can be combined with each other p62. RIP1ubiquitination is also associated with the NF-κB signaling pathway, and can activatecaspase-8to take part in apoptosis.Objective:In this study, we will further reveal the mechanism of human ovarian cancercell resistance, from p62affecting RIP1ubiquitination and participation in theactivation of NF-κB signal pathway.Methods:Human ovarian cancer cell line SKOV-3and their progeny cisplatin-resistantcell lines SKOV-3/DDP cells treated with cisplatin for the study. Experimentallysmall interfering RNA, the expression of p62protein was inhibited. The cell survivalrate was detected by MTT assay. Observing and taking photos of celluar morphologyby inverted optical microscope and a digital microscope system of automatic observation. The total exprssions of RIP1and p62proteins and the nuclear proteinNF-κB (p50), NF-κB (p65), phospho-IκBα (p-IκBα) and IκBα were detected byWestern Blot assay. p-IκBα/IκBα was evaluated to make sure the activation ofNF-κB signal pathway. Observed by immunofluorescence experiments NF-κB (p65)of the nuclear situation. By co-immunoprecipitation experiments, we test theexpression of RIP1ubiquitination in the form of changes, the activation of NF-κBsignal pathway and the cell survival rate.Results:1) Compared with SKOV-3, the cell survival rate slightly decreased and onlysome of the cells were oval and float in SKOV-3/DDP were treated by cisplatin12hand24h. Compared with SKOV-3, SKOV-3/DDP was significantly resistant.2) Compared with SKOV-3, p62protein in SKOV-3/DDP cell was increased.Under cisplatin, the expression of p62protein was decreased. RIP1proteinexpression in SKOV-3/DDP cells was high, and no obvious changes were seenunder cisplatin3) Compared with SKOV-3, the expressions of NF-κB (p50) and NF-κB (p65)were increased, p-IκBα protein and the ration of p-IκBα/IκBα were also increased,the nucleus colalization of NF-κB (p65) protein increases by immunofluence.4) Compared with SKOV-3, RIP1ubiquitination expression of K63was high,RIP1ubiquitination K48was low, and both were time-dependent byco-immunoprecipitation experiments.5) Inhibition of p62protein expression, RIP1ubiquitination expression of K63was significantly decreased, and RIP1ubiquitination K48was increased byco-immunoprecipitation experiments. Meanwhile the activation of NF-κB and thecell survival rate were all decreased.Conclusions:1) The proteins expression of p62and RIP1in SKOV-3and SKOV-3/DDP havedifferences, maybe related with the two cells line sensitivity to cisplatin.2) RIP1ubiquitin and the activation of NF-κB signal pathway in SKOV-3and SKOV-3/DDP have differences, maybe related with the two cells line sensitivity tocisplatin.3) p62regulates the NF-κB signaling pathway through impacting RIP1ubiquitination, which participate in cisplatin resistance of human ovarian cancer cell.