| BackgroundHepatocellular carcinoma(HCC)is one of the most common primary malignant tumors in the world.It has the characteristics of insidious onset,high incidence,rapid growth,strong invasiveness,high mortality,and poor prognosis,and there is still no effective treatment.For advanced liver cancer,in addition to the short-term efficacy of topical treatment,sorafenib is currently the only targeted drug for clinical treatment,but rapid drug resistance severely limits its clinical efficacy.HCC is less sensitive to chemotherapeutic drugs than other tumors,and its chemoresistance is one of the main reasons for the failure of drugs to treat HCC and its mechanism is extremely complicated.Therefore,it is urgent to deeply study the pathological mechanism of HCC and the new intervention targets behind chemotherapy resistance,and to develop a safe and effective new cancer treatment drug is of great significance.In recent years,metal drugs have achieved remarkable effects in the treatment of various cancers,and platinum complexes have been used in clinical applications to varying degrees.However,due to the occurrence of side effects such as drug resistance,it is the key to the current research of complex drugs to find complexes with strong anti-tumor activity,low toxicity and ability to overcome drug resistance.The physiological distribution of copper complexes,intracellular aggregation and inhibition of tumor cell growth have different degrees of difference with platinum complexes,which makes it possible for copper complexes to overcome drug resistance as antitumor drugs.In the previous study,our group found that the tri-pyridine copper complex can effectively bind to the tumor cell DNA and cleave the DNA into different components by the principle of oxidative cleavage,but it is not enough to significantly change the malignant biological behavior of the tumor cells.It may be that tumor cells can repair DNA damage in time,which is one of the causes of drug resistance.In order to further optimize the above tripyridine copper complex,our group first introduced tri-phenyl-phosphine(TPP)into the complex to obtain a new copper complex[Cu(ttpy-tpp)Br2]Br(expressed as CTB).TPP can increase the delocalized charge for the complex while its lipophilic properties facilitate aggregation onto the mitochondria.Inductively coupled plasma optical emission spectroscopy(ICP)confirmed that CTB has obvious mitochondrial targeting function and has good water solubility and DNA cleavage activity.However,the role and molecular mechanism of CTB targeting tumor cell mitochondria for antitumor activity is not fully understood,and further investigation is needed.The aim of this study was to clarify the anti-tumor effect of CTB on hepatocellular carcinoma and to elucidate the molecular mechanism of its action,providing a theoretical basis for the design and clinical application of mitochondria-targeted complex drugs in the future.MethodsThe research of this thesis is carried out in vitro and in vivo:1、In vitro researchHuman hepatoma cells(SMMC-7721 cells)were used in this research.Firstly,the most suitablconcentration of CTB was screened and the inhibition of tumor cells was detected by MTT.The effect of CTB on the mitochondrial structure of SMMC-7721 cells was examined by transmission electron microscopy,Janus green staining and JC-1 staining.Seahorse XF96 extracellular flow analyzer were used to measure the oxygen consumption rate and the external acidification rate for the determination of mitochondrial function.The effects of CTB on the apoptosis of hepatoma cells were detected by Annexin V-FITC/PI staining and TUNEL staining The accumulation of ROS in hepatoma cells was detected by DCFH-DA staining,and Transmission Electron Microscopy,MDC fluorescence and Plasmid Transfection detect the occurrence of autophagy in hepatoma cells.The effect of CTB on mitochondrial fission was investigated using mitochondrial fluorescent probes.The expression and activation of the target protein were detected by immunofluorescence double staining and Western blot.2.In vivo researchFour-week-old male nude mice(BALB/c-nu/nu)weighing approximately 18-22 g were purchased to establish a nude mouse xenograft model of human liver cancer SMMC-7721 cells.After successful construction,the caliper periodically measures tumor size and establishes a growth curve.The long diameter(a)and the short diameter(b)were measured by a vernier caliper,and the tumor volume was calculated by the formula,that isLthe volume(V)=a×b2/2.When the tumor reached an average size of 150 mm3,the mice were randomized according to the experimental needs(8 animals per group).The drug was injected intraperitoneally,and CTB was suspended in sterile PBS and injected three times a week for two weeks.At the end of the experiment,intact tumors,liver and other organs were isolated and weighed,tumors were photographed and weighed to calculate the tumor inhibition rate.The tumor tissue was partially excised and fixed in 4%paraformaldehyde for IHC/IF detection,or fixed in 2.5%glutaraldehyde for transmission electron microscopy,and the remainder was frozen in liquid nitrogen for Western blot detection.ResultsIn vitro studies had shown that CTB inhibited the growth of a variety of hepatoma cells in a time-and dose-dependent manner.Transmission electron microscopy,Janus green staining,JC-1 staining and other tests showed that CTB destroyed mitochondrial structure,leading to a decrease in mitochondrial membrane potential,and the results of extracellular acidification and oxygen consumption rate showed that CTB inhibited glycolysis and oxygen consumption of hepatoma cells.AnnexinV-FITC/PI staining and TUNEL staining showed that CTB induced apoptosis of hepatoma cells,accompanied by accumulation of ROS in hepatoma cells and mitochondrial translocation of p53 protein.Transmission Electron Microscopy and MDC fluorescence showed that CTB could increase the autophagy level of hepatoma cells.GFP-LC3 fluorescent reporter gene showed the activation of LC3-Ⅱ,while CTB activated autophagy AMPK/mTOR/ULK1 signaling pathway.Mitochondria and Lysosomes Fluorescence co-localization revealed the occurrence of mitophagy with an increase in the expression of the mitochondrial autophagy protein Parkin/PINK1.Further Mito-tracker Green fluorescence assay showed that CTB induced mitochondrial hypermutation accompanied by activation of mitochondrial protein Drpl.Moreover,Drpl specific inhibitor Mdivi-1 reduced the expression of mitophagy protein while inhibiting mitochondrial fission,impairing CTB-induced mitophagy.Final test results showed that the autophagy inhibitor,3-MA,inhibited CTB-induced mitochondrial dysfunction in hepatoma cells.Mdivi-1 inhibited CTB-induced mitochondrial translocation and apoptosis,showing that autophagy had a certain promotion effect on CTB-induced cell apoptosis.The results of in vivo studies showed that the tissue texture of tumor in nude mice transplanted with CTB was hard and whitish,and the tumor inhibition rate and growth curve showed that CTB inhibited the growth of tumor tissue in a dose-dependent manner.The results of TUNEL staining and the expression of apoptosis-related factors showed that CTB induced apoptosis of tumor tissues.At the same time,CTB increased the levels of autophagy marker proteins LC3-Ⅱ,Parkin and PINK1 in tumor tissues,and decreased the level of p62.The Drp1 inhibitor Mdivi-1 attenuated these effects,and inhibition of mitochondrial fission could affect the autophagy.ConclusionThe results of in vivo and in vitro studies fully demonstrated that CTB had good anti-hepatocarcinoma activity and was inseparable from its mitochondrial targeting properties.CTB induced mitochondrial dysfunction in hepatoma cells and induced mitochondrial apoptosis through ROS-dependent p53 mitochondrial translocation,thereby exerting its anti-tumor effect.CTB promoted the occurrence of mitoophagy through Drpl-dependent mitochondrial fission and played a vital role in promoting CTB-induced mitochondrial apoptosis.This study provided a certain experimental basis for the development of anti-hepatocarcinoma cell therapy drugs that target mitochondrial function. |
PDF Full Text Request