The Investigation Of Therapeutic Effects And Relevant Mechanisms In The CONPs Treated Renal Cancer

[Introduction]Clinically advanced renal cell carcinoma(RCC)patients no longer have surgery as a treatment option,and approximately onethird of RCC patients face relapse and metastasis after radical nephrectomy.Antiangiogenic drugs are standard treatments that target the vascular endothelial growth factor(VEGF)ligand itself or inhibit its receptors via proangiogenic receptor tyrosine kinase(RTK)inhibitors,such as sunitinib.Unfortunately,fewer thanone-fifth of RCC patients who are refractory to antiangiogenic healing and show tumour progression and nearly all remaining patients eventually develop resistance after receiving one or more of these therapies.Because the discontinuation of sunitinib therapy in RCC patients results in the rapid reappearance of angiogenesis,developing new drugs for RCC or sunitinib-resistant RCC therapy is necessary.With the rapid development of nanoscience,the unique pharmacological effects of various types of nanomedicines have been explored,many of which have been investigated in clinical trials and used in clinical applications.Among these nanomedicines,inorganic nanomedicines have been widely studied and have shown ideal pharmacological effects in clinical applications.The nanoparticle Fe3O4 has been approved by the USA Food and Drug Administration(FDA)and is used for the treatment of anaemia.Recently,Fe3O4 nanoparticles were shown to activate macrophages and thus may be used in cancer immunological therapy.Additionally,silver nanoparticles have been used in anti-infective therapy for obstetric gynaecological diseases and burns.Other inorganic nanomedicines,such as Zn O and gold,have also shown potential pharmacological effects,including anti-tumour effects.In our previous research,cuprous oxide nanoparticles(CONPs)showed ideal anti-tumour effects and low systemic toxicity.Copper chaperone proteins,which regulate the endocellular dosage and transport of copper,also play crucial roles in the progression of cancer.The interaciton between nanomedicine and caopper chaperone proteins and their detailed mechanisms remain unknown.[Objective]Investigate the effects of CONPs on the biological characteristics of renal cell carcinoma(RCC),and verified it in vivo experiments.On the basis of positive pharmacodynamics,investigate the molecular mechanism of CONPs inhibit the progression of RCC,and research the targets of CONPs suppression of RCC proliferation and induction of its apoptosis.[Methods]1.The RCC cell viability and the effects on proliferation were determined by CCK8 methods,the effects of CONPs on RCC migration and invasion were tested by the transwell experiments costed with or without Matrigel.Cell apoptosis was assessed using the V-FITC/PI Apoptosis Detection Kit and analysed on the flow cytometry analyzer.The cell cycle was checked by the cell cycle staining kit on the flow cytometry analyzer.2.The transcriptional level of targeted RNA were detected by RNA extraction and quantitative real-time-PCR(q RT-PCR).The translational level of targeted proteins were determined by the Western blot assay(WB).The lentivirus-based short hairpin RNAs(sh RNAs)were designed and synthesized to silence targeted protein to testify drug targets of CONPs.3.The subcellular structures of RCC cells incubated with CONPs were observed by a Transmission electron microscopy(TEM)system.Total copper content in RCC cells were detected by inductively coupled plasma mass spectrometry(ICP-MS)based Cu absorption assay.The intracellular reactive oxygen species(ROS)production were measured by intracellular oxidative activity to verify the activation of ER stress in RCC.4.The anti-RCC effects were verified by the vivo tumour xenograft experiments.The subcutaneous tumour tissues were stripped and performed TUNEL and KI67 experiments.To step further verify the function of CONPs on the proliferation and apoptosis of RCC in vivo experiments.[RESULTS]1.CONPs inhibit RCC cell proliferation,and induce RCC cell cycle arrest at the S and G2 phases.In addition,CONPs suppress RCC cell migration and invasion.2.TEM found the CONPs make the ER of RCC cells drastic swelling.CONPs activate ER stress by promoting ROS production and cytosolic Ca2+ levels,which was detected by Flow Cytometry.The upregulation of ROS levels and cytosolic Ca2+concentration was the initiation of ER stress.3.CONPs induce intercellular copper accumulation,and copper chaperone proteins evoke anti-tumour effects.CONPs induce RCC cell death via ER stress-regulated apoptosis,and WB experiments verified the relevant protein were activated on the athways of ER-stress and apoptosis.4.CONPs inhibit RCC tumour development in mice,and CONPs may target AXL and MET,thus restoring sunitinib responsiveness in RCC cells with SR.In addition,CONPs activate ER stress,apoptosis and sunitinib-resistant reversal in vivo.[CONCLUSION]In this study,we systemically explored the pharmacological effects and mechanism of inorganic CONPs nanomedicines on the inhibition of RCC and sunitinib-resistant RCC.Multiple assays were utilized to illustrate both the in vitro and in vivo effectiveness of CONPs,and we explored the importance of the copper chaperone proteins ATOX1 and CCS to the pharmacological performance of CONPs.We showed that CONPs could influence ATOX1 and CCS function,disturb intercellular copper trafficking,and release cuprous ions,which led to the accumulation of total elemental copper,ER stress activation,and RCC apoptosis and proliferation inhibition.CONPs could also elevate ROS and calcium levels in RCC cells directly by influencing ER stress.CONPs displayed strong anti-RCC properties with little renal and liver toxicity in vitro and in vivo and decreased the levels of AXL,MET,AKT,and ERK to overcome sunitinib resistance.These investigations should facilitate the development of CONPs as a novel therapeutic approach for the clinical treatment of RCC and advanced RCC showing resistance to sunitinib,which is paramount to further improving patient outcomes.