Mechanism Of The Multifunctional Protein P62 In Drug Resistance In Human Ovarian Cancer Cells

Ovarian cancer, the deadliest gynecological malignancy, is typically treated in its advanced stages with cytoreductive surgery and platinum-based chemotherapy. Cisplatin (cis-diamminedichloroplatinum) is widely used as a chemotherapeutic agent for ovarian cancers. However, cisplatin resistance limits its use in cancer patients.Recent studies have shown that cisplatin-induced apoptotic signaling can occur through endoplasmic reticulum (ER) stress, suggesting that the ER is a cytosolic target of cisplatin. When cells were in endoplasmic reticulum stress, the unfolded protein response (UPR) was triggered by the accumulation of misfolded proteins, and the classic endoplasmic reticulum associated protein degradation (ERAD) and ER-activated autophagy, ERAA) pathway were involved in the degradation of the misfolded proteinsThe ubiquitin-binding protein, p62 (sequestosome 1) is multifunctional; it promotes survival-critical signals, including proliferation, differentiation and induction of anti-apoptotic genes. The protein p62 has unique features: an N-terminal Phox, and a Bem1p domain (capable of self-oligomerization), and a C-terminal ubiquitin-associated domain, which interacts with ubiquitinated proteins. These properties imply p62's involvement in aggregate formation. In autophagy-impaired mice and flies, additional p62 loss is linked to reduced formation of ubiquitin inclusions. p62 is selectively degraded by autophagy. It can act as a receptor or adaptor for ubiquitinated substrate autophagy, which is mediated through microtubule-associated protein 1 light chain 3 (LC3). The LC3 is drawn to phagophore/isolation membranes and remains coupled to completed autophagosomes. Some studies showed that p62 may affect ubiquitinated proteins'linkage to autophages through LC3/GABARAP, thus maintaining cell homeostasis and survival.Some studies have shown that Keap1-Nrf2 signal pathway was related to drug resistance of cancer cells. p62 may be competitive binding with Keap1, thus Nrf2 can translate into the nucleus, activating the Nrf2 target gene transcription. Some studies suggest that the KIR domain of p62 directly binds with the Kelch repeat domain of Keap1, blocking the combination of Keap1 and Nrf2, the Keap1 was degraded by the autophagy pathway and Nrf2 was activated. Therefore, p62 acts as a key regulator in drug resistance through regulating autophagy and/or Keap1-Nrf2-ARE signal pathway.Here, we chose cisplatin-sensitive SKOV3 and their cisplatin-resistant clones SKOV3/DDP as the in vitro model to examine the mechanism of p62 in cisplatin resistance in human ovarian cancer cells. This maybe a new route of cancer therapy.Methods(1) Base on the gene sequence of p62 (GenBank: NM₀03900) and the RNAi desine principles, pGCsilencerTM U6/Neo/GFP/RNAi vector was used to construct the recombinant plasmid si-p62. The level of p62 mRNA and protein expression was analyzed by RT-PCR and Western blot.(2) Cell viability was detected by MTT assay. Apoptotic nuclear changes were assessed with Hoechst 33258. The expression level of ER stress related proteins (including Grp78, GADD153, Cleaved Caspase-4), ubiquitinated proteins, and Cleaved Caspase-3 were detected by Western blot.(3) The level of p62 mRNA and protein expression was analyzed by RT-PCR and Western blot. The colocoalisation of p62 and LC3 was detected by fluorescence microscopy. Photomicrographs of human ovarian cancer cells were by epresentative transmission electron microscopy. The accumulation of LC3 was analysed by western blot.(4) Inhibited autophagy or proteasome pathway by 3-MA or LAC. Cell viability was determined by MTT assay. The expression level of ER stress related proteins (including Grp78, GADD153, Cleaved Caspase-4), ubiquitinated proteins were detected by Western blot.(5) Inhibited the expression of p62 by RNAi. Cell viability was determined by MTT assay. The expression level of ER stress related proteins (including Grp78, GADD153, Cleaved Caspase-4), ubiquitinated proteins were detected by Western blot. Apoptotic nuclear changes were assessed with Hoechst 33258. Apoptotic cell populations were quantified by flow cytometry analysis.(6) Oxidative damage model of human ovarian cancer cells was produced by H₂O₂. Cell viability was determined by MTT assay. Apoptotic nuclear changes were assessed with Hoechst 33258. The expression of Cleaved Caspase-3 was detected by Western blot.(7) The colocoalisation of p62 and Keap1 was detected by fluorescence microscopy. The expression level of Keap1 and Nrf2 were detected by Western blot. The level of GSTP1, ALDH1A7 mRNA expression was analyzed by RT-PCR.Results(1) There was significant on cell viability in cisplatin-sensitive SKOV3 cells and cisplatin-resistant SKOV3/DDP cells. Results showed that SKOV3/DDP cells were more resistant than SKOV3 cells. Cisplatin induced the ubiquitinated proteins and the endoplasmic reticulum stress proteins, including Grp78, GADD153, Cleaved Caspase-4 upregulated, most cells were apoptotic. The same concentration of cisplatin didn't not affect the associated protein expression in SKOV3/DDP cells,and Very few cells were apoptotic.(2) Cisplatin-resistant SKOV3/DDP cells express much higher p62 levels than do cisplatin-sensitive SKOV3 cells in mRNA and protein level. Cisplatin induced the p62 protein decreased.(3) Cisplatin induced the colocalisation of p62 and LC3 in SKOV3/DDP cells. This indicated that cisplatin induced autophagy in SKOV3/DDP cells.(4) Inhibited autophagy pathway by 3-MA, the expression of p62, ubiquitinated proteins, ER stress related proteins, apoptosis related proteins were up regulated in SKOV3/DDP cells. Inhibition of proteasome pathway by LAC, the expression of ubiquitinated proteins were slightly up regulated, but the level of p62, ER stress related proteins, and apoptosis related proteins showed no significant changes in SKOV3/DDP cells. These results indicated that p62 was degradated by autophagy.(5) We constructed the recombinant plasmid si-p62 and transfected to SKOV3/DDP cells. The results showed that cisplatin decreased the survival rate of SKOV3/DDP cells transfected with si-p62, and significantly up regulated the expression of ubiquitinated proteins, ER stress related proteins, apoptosis related proteins.(6) Both cisplatin and H₂O₂ increased the level of ROS in SKOV3 cells, but not in SKOV3/DDP cells.(7) Cisplatin-resistant SKOV3/DDP cells express much higher p62 levels than do cisplatin-sensitive SKOV3 cells, and H₂O₂ induced p62 protein levels decreased gradually while p62 mRNA transcripts remained constant in SKOV3/DDP cells. H₂O₂ induced the colocalisation of p62 and Keap1, decreased the protein level of p62 and Keap1, and increased the level of Nrf2 in nuclus of SKOV3/DDP cells. In addition, the level of GSTP1, ALDH1A7 mRNA were up regulated. These results indicated that H₂O₂ actived the Keap1-Nrf2-ARE signal pathway.(8) Inhibited the expression of p62 by RNAi, SKOV3/DDP cells were more resentive. The levels of GSTP1 and ALDH1A7 mRNA were no significant changes in SKOV3/DDP cells transfected with si-p62, and the apoptotic cells were significantly increased. This indicated that p62 was involved in Keap1-Nrf2-ARE signal pathway.ConclusionsThis research chose cisplatin-sensitive SKOV3 and their cisplatin-resistant clones SKOV3/DDP as the in vitro model to examine the mechanism of p62 in cisplatin resistance in human ovarian cancer cells. Our results indicated that the difference of apoptosis medicated by ER stress and p62 protein level indued by cisplatin. We found cisplatin could induce the autophagy in SKOV3/DDP cells by different methods. In addition, our results indicated that p62, the Multifunctional protein, could target ubiquitinated proteins for degradation through autophagy, thus maintaining cell homeostasis. Inhibition of the p62, the expression of ubiquitinated proteins, ER stress related proteins, apoptosis related proteins were significantly up regulated.Meanwhile, we found cisplatin could induce oxidative damage in SKOV3 cells, but not in SKOV3/DDP cells. In the cell model produced by H₂O₂, p62 can activate the Keap1-Nrf2-ARE signaling pathway and induce the expression of antioxidant genes, reducing the level of intracellular ROS. Therefore, the cisplatin-resistant mechanism of SKOV3/DDP cells may be combined with the p62 targeted ubiquitinated proteins for degradation through autophagy, maintaining cell homeostasis; and regulated the Keap1-Nrf2-ARE signaling pathway, activating the expression of antioxidant genes.This study first discussed the role of p62 which a multifunctional protein in cisplatin-resistance in human ovarian cancer cells. On the one hand, p62 target ubiquitinated proteins for degradation through autophagy, thus maintaining cell homeostasis. On the other hand, p62 can regulate Keap1-Nrf2-ARE signaling pathway, activate the expression of anticridant genes, decreasing the levels of intracellular ROS. Our results showed that, p62 as a multifunctional protein, may be regulate the cisplatin resistance in human ovarian cancer cells. More in-depth studies on p62, maybe provide a new route of cancer therapy.