Studies On Anti-tumor Effect And Mechanism Of Bufalin And Its Liposome Preparation

IntroductionThe global cancer statistic had reported that the incidence and mortality of cancer were increased rapidly.Cancer is responsible for about 18.1 million new cases and an estimated9.6 million deaths worldwide in 2018.China accounted for nearly 23.7%of new cases,while the number of deaths was 35.8%,which is the highest in the world.The 2020 Cancer Statistics in American reported that from 1991 to 2017,total mortality of cancer in the United States continued to decline by 29%.However,the five-year survival rate of cancers,including lung cancer,esophageal cancer and liver cancer with high incidence in China,is generally low and the mortality rate is still high.The prevention and treatment of cancer is still a big problem in China.Now the cancer treatment is mainly based on the surgery,and partially based on radiotherapy and chemotherapy.Radiotherapy can kill the tumor cells and inhibit the tumor cell proliferation in a short time.Chemotherapy has a good effect on some solid tumors.However,because of its low specificity,the normal human cells are inhibited,and the immune system is destroyed in the meanwhile.Therefore,it is an urgent scientific problem to find and develop effective anti-tumor drugs with low toxicity.Chan Su were extracted from the skin and parotid venom glands of the toad Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider.“Compendium of Materia Medica”had recorded that Chan Su“cures all the malignant swelling”,which had a long history for the tumor treatment.Bufalin is one of the main active components of Chan Su.Some studies have shown that bufalin has a variety of pharmacological activities,including cardiotonic,anti-viral,immune-regulation and especially anti-tumor effects.At present,studies have proved that bufalin has certain inhibitory effect on many solid tumors,such as colon cancer,liver cancer,melanoma and glioma cancer.However,the mechanism underlying cell apoptosis induced by bufalin had not yet been explicated.Moreover,the poor solubility and strong cardiotoxicity restrict its clinical application.At present,studies on nano-prepations had showed that liposomes have the advantages of increasing drug stability,improving water solubility,reducing drug toxicity and long-term targeting effect.The surface modification of polyethylene glycol（PEG）on the liposomes could increase the steric hindrance and the stability of liposomes,promote the liposomes to cross the blood-brain barrier and increase the accumulation of liposomes reaching brain tissue.It can prevent from being swallowed and cleared by reticular endothelial cells under the action of plasma proteins,and has a good prospect to be used in drug targeted delivery system.On the basis of my master’s study,the proapoptotic effect of bufalin on Hep G2,HCT116,HCT8 and U251 cells were evaluated in this study.Drug affinity responsive target stability（DARTS）technolgy,combining with SDS-PAGE,silver stain,LC-MS/MS identification and proteomics information analysis,were used to identify and verify the target proteins directly affected by bufalin on U251 cells,and the mechanisms of bufalin-induced apoptosis in U251 cells were studied.Moreover,the physicochemical characterization,biological safety and anti-tumor activity of the prepared bufalin loaded PEGylated liposomes（BF/PEG-LP）were investigated,which provided experimental basis and data support for its future clinical application in anti-tumor therapy.Methods1.Study on the antitumor activity of bufalin-induced apoptosisGuided by the theory of cell biology,the viability of 25,50 and 100 n M bufalin-treated Hep G2,HCT116,HCT8 and U251 cells were evaluated by CCK-8 assay,colony formation assay and flow cytometry.The REDOX equilibrium was measured by the level of reactive oxygen species（ROS）and glutathione（GSH）,and the ion homeostasis was estimated by Na⁺/K⁺-ATPase activity and intracellular calcium ion(Ca²⁺)level.Mitochondrial membrane potential（MMP）measured by JC-10 staining and the level of adenosine triphosphate（ATP）were detected to evaluate the mitochondrial function of Hep G2,HCT116,HCT8 and U251 cells.Flow cytometry was also used to test the cell cycle of Hep G2,HCT116 and U251 cells.Western blot was used to determine the expression of apoptosis（Caspase 3,Cleaved Caspase 3 and Cytochrome C）and DNA damage（γ-H2A.X,H2A.X,ATR,p-ATR,Chk1,p-Chk1 and c-myc）related proteins in cancer cells.Quantitative real-time polymerase chain reaction（q RT-PCR）was used to detect the m RNA level of DNA damage checkpoints（ATM,ATR,Chk1,Chk2,CDC25A and CDK2）in S-phase arrest,which was detected to preliminarily elucidate the antitumor activity and pro-apoptotic effect of bufalin on tumor cells in vitro.2.Target identification of bufalin in inhibiting glioma U251 cellsUnder the guidance of the pharmaceutical analysis theory,the target proteins directly affected by bufalin（100 and 1000μM）on U251 cells were identified and verified,and the mechanisms of bufalin-induced apoptosis in U251 cells were studied.Using the DARTS-PAGE technology,combined with silver stain,LC-MS/MS identification,proteomics information analysis,molecular docking and Western blot experiments,the direct target proteins of bufalin inhibiting the activity of U251 cells were identified and validated.Moreover,the protein expression level of Annexin A2 was evaluated by Western blot in bufalin-induced U251 cell.The impact of Annexin A2 on bufalin-induced U251 cell apoptosis was studied by si RNA technology.After treatement of bufalin（25,50,and 100n M）for 12 h,Mitotracker red CMX Ros,Annexin V-FITC and Hoechst33342 were co-stained to detect early apoptosis and mtichondrial biological activity of U251 cells by confocal.After drug treatement for 24 h,the mitochondrial morphology was evaluated by Mitotracker green staining,the changes of intracellular ultrastructure were observed by transmission electron microscopy（TEM）,and the surface volume ratio（Rsv）,volume density（Vv）,surface density（Sv）and number density（Nv）of mitochondria were calculated according to stereoscopic theory.The expression levels of cell division/fusion protein DRP1in the cytoplasm and mitochondria were detected by Western blot,so as to evaluate the damage of bufalin to the mitochondrial structure and function of U251 cells.Preconditioning with the DRP1 protein inhibitor mdivi-1（20μM）for 2 h was used to evaluate the effect of DRP1 protein on bufalin-induced mitochondrial structural snd functional dysfunction and apoptosis of U251 cells.3.Studies on the anti-glioma effects of BF/PEG-LPUnder the guidance of pharmacy theory,the physical and chemical properties,biological safety and anti-glioma activity of BF/PEG-LP in master’s study were investigated.To characterize the physicochemical properties of bufalin liposomes,the surface morphology of bufalin liposomes was observed by TEM,the characteristic peaks of BF/PEG-LP were characterized by FT-IR,and the stability of BF/PEG-LP in different p H environment was studied by in vitro release test.The safety of membrane preparation materials of BF/PEG-LP was tested by RBC hemolysis assay and CCK-8 assay.The half lethal dose(LD₅₀)of bufalin liposomes was compared with that of bufalin liposomes by acute toxicity test in mice.The difference of tissue distribution between bufalin and bufalin liposomes（1.0 mg/kg）was studied in rats.The effects of bufalin liposomes（0.5,1.0 and2.0 mg/kg）on general behavior,autonomic activity and body coordination were examined in mice.Moreover,CCK-8 assay was used to detect the anti-tumor activity of BF/PEG-LP（0-1000 n M）in vitro and transplantations of glioma U251 in nude mice were used to investigate the inhibitory effect of BF/PEG-LP（0.5,1.0 and 2.0 mg/kg）in vivo.Results1.Bufalin induced cell DNA damage and mitochondrial dysfunction,and had a significant killing effect on a variety of tumor cells,among which U251 and HCT116were more sensitive.Median inhibition concentration(IC₅₀)of bufalin on Hep G2 cells at 12,24 and 48 h were 201.10±15.94,143.85±13.74 and 33.45±0.39 n M,those of HCT8 cells were 111.02±11.41,76.30±13.14 and 23.67±7.84 n M,those of U251 cells were 190.48±6.67,52.31±4.77 and 25.57±9.53 n M,and those of HCT116 cells were 155.36±13.38,32.86±7.06and 9.00±4.66 n M,respectively.The 48 h IC₅₀ values for Hep G2,HCT8,HCT116 and U251 cells were all lower than 35 n M,indicating that bufalin could inhibit the growth of a variety of tumor cells.The 24 h IC₅₀ value for HCT116 and U251 cells was as low as 33-55 n M,indicating that bufalin had a more significant killing effect on these two tumor strains.Moreover,bufalin treatment could inhibit cell proliferation and colony formation,and induce apoptosis in Hep G2,HCT8,U251 and HCT116 cells.The increased intensity of ROS indicator and the decreased levels of GSH content in Hep G2,HCT116,HCT8 and U251 cells demonstrated that bufalin treatment（25,50 and100 n M）caused oxidative stress.Moreover,bufalin treatment inhibited the activity of Na⁺/K⁺-ATPase and increased intracellular Ca²⁺level of Hep G2,HCT116 and HCT8 cells,which triggered the ion imbalance,deficiency of MMP and dysfunction of mitochondria.Consequently,Cytochrome C was up-regulated and Caspase 3 signal pathway was activated,which induced cell apoptosis.Simutaneously,bufalin could induce the phosphoration of H2A.X,significantly up-regulate phospho-histone H2A.X（γ-H2A.X）,phospho-ATR（p-ATR）and phospho-Chk1（p-Chk1）and down-regulate H2A.X,ATR and Chk1 in Hep G2 cells.In HCT116 and U251cells,bufalin could significantly up-regulate the expression levels of p-Chk1,and down-regulate the protein expression levels of Chk1,which indicating that bufalin could induce DNA damage in vitro.Moreover,the m RNA expression levels of s-phase DNA damage checkpoints,including ATR,Chk1,CDC25A and CDK2 to transmit DNA damage signals.Meanwhile,the protein expression levels of G1/S transcription factor c-myc was up-regulated,and directly arrested at S-phase in Hep G2,HCT116 and U251 cells in vitro,which finaly caused cell apoptosis.2.Bufalin directly regulates Annexin A2 and DRP1 proteins to induce apoptosis in U251 cells.In order to further clarify the mechanism of anti-tumor activity of bufalin on U251cells,total proteins of U251 cells were co-incubated with bufalin.Using DARTS-PAGE technology,combining with silver staining and LC-MS/MS,258 proteins（Unused≥1.3）potential direct target proteins of bufalin acting on U251 cells were screened out.Using FUNRICH software,Geno Othology（GO）enrichment analysis found that the differential proteins mainly exist in the cytoplasm,lysosome,nucleus and mitochondria,which was involved in protein metabolism,cell growth/maintain,mitochondria transportation and carbohydrate metabolism on the necessary key steps in the process of glioma cell proliferation,through biological signaling pathways such as protein metabolism,gene expression and translation.Annexin A2,TUBb,DRP1,HSPA8 and HSPA9 in the bufalin group were only partially unenzymized compared with the control group by DARTS-PAGE experiments,through the co-incubation of representative differential proteins and bufalin.Molecular docking experiments simulated binding models of proteins and small molecule drugs,showing that Annexin A2,TUBb,DRP1,HSPA8,and HSPA9 can directly bind bufalin to form hydrogen bonds through intermolecular interactions.DARTS-Western blot experiment was conducted on the total protein of U251 cells and bufalin complex,further verifying that bufalin could bind Annexin A2 and DRP1 proteins directly in U251 cells.After treatment with 100 n M bufalin for 24 h,both Annexin A2 and DRP1 proteins underwent mitochondrial translocations in U251 cells.Using si RNA technology,it was demonstrated that after the down-regulation of the expression level of Annexin A2 protein,the cell survival rate of U251 cells after bufalin administration significantly decreased,which indicating the sensitivity of U251 cells to bufalin increased.Cell function experiments showed that compared with control group,the intracellular ultrastructure was destroyed,and the mitochondria network was sparse in bufalin-treated U251 cells.Moreover,Nv was significantly increased,while Vv,Sv and Rsv were significantly decreased,indicating that bufalin could disorder the mitochondrial structure and function of U251 cells,resulting in more and smaller mitochondria,resulting from the disruption of the mitochondrial division/fusion balance by DRP1 protein,which indicating that bufalin had strong anti-glioma effect in U251 cells in vitro.The DRP1protein inhibitor Mdivi-1 can partially improve the mitochondrial structure abnormality caused by bufalin in U251 cells,protect the mitochondrial function and reduce the proportion of apoptotic cells.3.BF/PEG-LP have stable physical and chemical properties,and its safety and anti-glioma effect are better than bufalin.BF/PEG-LP were homogeneous and spherical,with particle size of 119.03±24.62 nm.The characteristic peaks at 3495 cm^-1 and 3436 cm^-1 were the stretching vibration of N-H of the amide bond,and that of 1079 cm^-1 is the stretching vibration of the ether bond C-O-C in BF/PEG-LP.BF/PEG-LP was more stable in the PBS buffer with p H7.4 than that of p H5.0.The blank liposomes have no abvious hemolysis and no toxicity to U251,Hep G2 and HCT116 cells,which explicated the safety of preparation materials of BF/PEG-LP.Moreover,the hemolysis rate（HR%）of BF/PEG-LP was significantly lower than bufalin,while the hemolysis of the bufalin samples was obvious.The general pharmacological research showed that BF/PEG-LP had certain impacts on the function of the general behavior,independent activities and coordinate excises in mice after a week of administration compared with the control group.For the acute toxicity test,the medium lethal dose(LD₅₀)values of bufalin and BF/PEG-LP in mice were 0.156 and 3.03 mg/kg by intravenous injection,respectively.Tissue distribution profiles showed that an obvious higher concentration was found in brain tissue and a greater reduction was detected in heart tissue when using BF/PEG-LP than that of bufalin.CCK-8 test showed that BF/PEG-LP could inhibit the proliferation of U251 cells in vitro significantly,which was stronger than that of bufalin group.Tumor xenograft experiments showed that BF/PEG-LP significantly inhibited the growth of xenografted U251 cells in vivo.And the inhibitory effect of BF/PEG-LP was stronger than that of the same dose bufalin group and DDP group.Conclusion（1）Bufalin has a broad spectrum of anti-tumor effects,among which the killing effects on HCT116 and U251 cells are stronger.As the main active component of toad venom,bufalin could inhibit the proliferation of a variety of tumor cell lines.Moreover,it is more sensitive to HCT116 and U251 cells.This proapoptotic effect might be closely related to mitochondrial oxidative stress and cell cycle arrest at S phase.（2）The apoptosis of U251 cells induced by bufalin was closely related to its direct target proteins Annexin A2 and DRP1.Annexin A2 and DRP1 were first identified as potential direct targets of butfalin on U251 cells.After bufalin treatment,Annexin A2 and DRP1 proteins underwent mitochondrial translocation in U251 cells.After down-regulating the protein expression of Annexin A2,the toxicity of bufalin to U251 cells was enhanced.Bufalin can directly regulate the mitochondrial division/fusion balance through DRP1protein,resulting in mitochondrial structure damage and dysfunction,and inducing apoptosis of U251 cells.（3）Compared with bufalin,BF/PEG-LP have better proprietary compatibility,higher safety and stronger anti-tumor efficacy.BF/PEG-LP exhibited the lower hemolysis,higher LD₅₀ in mice,increased enrichment in brain,decreased accumulation in heart and better anti-tumor properties on U251 cells in vitro and in vivo than bufalin.In summary,this study suggests that bufalin and its liposomes are valuable for further research and development as an anti-tumor drug.