Studies On Oxoaporphine Alkaloid Metal Complexes To Kill Bladder Cancer Cells Via Multi-Mechanisms Of Autophagy,Microtubule Disruption And Immunogenic Cell Death

Cancer has exceeded the cardiovascular diseases as the main cause of death,which caused an estimated 10 million deaths worldwide in 2020 alone.Bladder cancer is one of the most common malignancies of the urinary system,causing approximately 250,000deaths per year.Despite the recent advances in treatment,the current chemotherapeutic drugs used to treat bladder cancer are still far from meeting demand of the bladder cancer treatment,and there is an urgent demand to find new anti-bladder cancer drugs.As platinum drugs produced serious toxic side effects and drug resistance in clinical applications,it is urgent to find more non-platinum drugs with high efficiency and low toxicity.Oxoaporphine alkaloids are a kind of active ingredients of Traditional Chinese medicine with extensive biological activities.Because of their molecular planar structure and unique pyridine nitrogen and carbonyl oxygen donor,they can coordinate with metal ions to form stable metal complexes.The purpose of this dissertation is utilization of active ingredient in traditional Chinese medicine（TCM）oxoaporphine and its derivatives as ligand to coordinate with potentially active metal center and to achieve efficient low toxicity of metal-based antitumor drugs.And then,the multiple mechanisms of action of these complexes on bladder cancer T-24 cells were carried out,and their antitumor activity and immunotherapy effects were investigated.The main research contents were as following:1.Eight kinds of oxoaporphine alkaloids were synthesized by means of Suzuki–Miyaura coupling and intramolecular oxidative cross dehydrogenation coupling.Most compounds inhibited the growth of T-24 cells in vitro.Particularly,compound 4B displayed the most potent activity with an IC₅₀ value of 0.5μM,which was 19-fold more potent than the parent compound 4.The substitution at C3-position of oxoaporphine core by-NO₂ significantly enhanced the anticancer activity,but compound 4A bearing a-NO₂ on the 9-positon did not show antitumor activity,which offered reference for further optimization of oxoaporphine derivatives.Mechanism studies indicated that compounds 4 and 4B induced cell cycle arrest at G2/M phase;however,compound 4C induced cell cycle arrest at the S phase.Compounds 4,4B and 4C reduced cell migration and induce DNA damage.Increase of mitochondrial ROS/Ca²⁺and decrease of MMP,accompanied by activation of caspase-3/9,were observed in T-24 cells after exposure to compounds 4,4B and 4C,suggesting that the mitochondrial pathway was involved in the induced apoptosis.Moreover,compound 4B effectively inhibited tumor growth in a mouse xenograft model bearing T-24.Compound 4B is a potential anti-bladder cancer leading compound.2.Four arene osmium（Ⅱ）and ruthenium（Ⅱ）complexes with ligands（4F or 4G）were synthesized and characterized.Among these metal complexes,4G-Os displayed the most potent activity.Mechanism studies showed that 4G-Os promoted tubulin polymerization into microtubules,and disrupted the microtubule skeleton network,which caused cell cycle arrest in G2/M phase.4G-Os inhibited T-24 cells growth through the mitophagy mechanism.It stimulated both the endoplasmic reticulum and mitochondria to induce endoplasmic reticulum stress（ERS）and mitochondrial dysfunction.It induced ERS through the PERK-CHOP pathway,thereby initiating PINK1/Parkin mediated mitophagy,and ultimately triggered the mitochondrial-mediated apoptosis.4G-Os caused severe impairment of mitochondrial bioenergetic function including mitochondrial depolarization,ATP levels and ATPase activity decrease,and mitophagy occurrence.In vivo antitumor experiments showed that 4G-Os exhibited strong antitumor activity and good in vivo safety.Overall,4G-Os mainly inhibited T-24 cells growth by inducing cell cycle arrest and mitophagy.3.Ten lanthanide complexes with ligands（4F or 4G）were synthesized and characterized.These lanthanide complexes showed good antitumor activity against T-24,SK-OV-3 and Hep G-2 cells,especially for T-24 cells and Hep G-2 cells,their IC₅₀ values were within 10μM range.These lanthanide complexes could significantly increase intracellular ROS and Ca²⁺levels,further destroyed mitochondrial membrane potential and activated caspase-3.Western blotting analysis showed that these lanthanide complexes up-regulated the expression of LC3B-II,suggesting the increase of autophagosome.Meanwhile,fluorescence staining study further confirmed the occurrence of autophagy.In brief,these lanthanide complexes caused caspase-3 and autophagy mediated cell death.4.Complexes of[NiL₂（NO₃）₂]（Complex 1）,[CuL₂（NO₃）₂]（Complex 2）and[Zn L₂（NO₃）₂]（Complex 3）with 1-trifluoroethoxyl-2,9,10-trimethoxy-7-oxoaporphine（L）ligand were synthesized and characterized.Complexes 1-3 can effectively target microtubule.Molecular docking revealed that complexes 1-3 bonded withβ-tubulin interacting with Arg278,Arg284 or His229 residues through hydrogen bond orπ-πinteraction.Complexes 1-3 showed good affinity toβ-tubulin by the surface plasmon resonance technology（SPR）and cellular thermal shift assay（CETSA）.Tubulin polymerization analysis suggested that complexes 1-3 promoted tubulin polymerization into microtubules,and disrupted the microtubule skeleton network,which caused cell cycle block in G2/M phase.In addition,complex 1 showed greater tumor growth inhibition efficacy than cisplatin in mice bearing T-24 xenograft,and good in vivo safety profile.All results suggested that complexes 1-3 exhibited high antitumor activity by disrupting microtubule function.5.Zn1 and Zn2 are Zn-based complex that activate antitumor immune response by inducing immunogenic cell death（ICD）.Complexes of Zn1 and Zn2 with 1,2,9,10-tetramethoxy-7-oxoaporphine（oxoglaucine/L¹）and 1-trifluoroethoxyl-2,9,10-trimethoxy-7-oxoaporphine（L²）were synthesized and characterized.Compared to Zn1,Zn2 bearing the-OCH₂CF₃ effectively caused ROS overproduction in early phase and subsequently endoplasmic reticulum stress（ERS）.Intensive ERS caused Ca²⁺to be released from ER and further promoted ER-mitochondrial Ca²⁺flux,which finally triggered mitochondrial dysfunction.The damage-associated molecular patterns（DAMPs）of CRT,HMGB1 and ATP were occurred in T-24 cells triggered by Zn1 and Zn2.The vaccination studies demonstrated that Zn1 and Zn2 efficiently suppressed the distant tumors growth,indicating the effective immune response by the induction of immunogenic cell death.The elevated CD8⁺cytotoxic T cells and decreased Foxp3⁺cells in vaccinated mice further verified the conclusion.Moreover,improved mice survival rate was observed after treatment of Zn1 and Zn2.Taken together,ICD induced by specific ROS-induced ER stress and mitochondrial dysfunction worked together to exhibit a strong tumor inhibition effect.