Research On The Anti-tumor Efficacy And Mechanism Of Hydrophilic Realgar Nano Preparations

Background:The application of arsenic has a history of 2000 years.In the practice of modern medicine,arsenic trioxide(AS2O3)combined with all-trans-retinoic acid(ATRA)has become the front-line therapeutics strategy for acute promyelocytic leukemia(APL),AS2O3 has also been approved in the treatment of advanced liver cancer.However,AS2O3 can easily lead to acute toxicity such as acute hemorrhage,gastrointestinal dysfunction,and adverse reactions such as gastrointestinal tract,skin and liver.Therefore,realgar,a mineral drug of arsenic in traditional Chinese medicine resources,has also received clinical attention.Realgar is a chemical component of arsenic tetrasulfide(AS4S4)and has been used in the treatment of leukemia in China since the 1960s.Compound Realgar Natural Indigo Tablet,with realgar as the main component,combined with ATRA show no inferior effect in APL than AS2O3,indicating that realgar could be an alternative to AS2O3 to avoid the side effects of As2O3.However,a major obstacle in the application of realgar is its poor water solubility,which leads to low bioavailability.In order to obtain the blood arsenic concentration required for treatment in clinical practice,realgar was often intaken massively,causing health risks and economic burden to patients.On the other hand,because of the extremely low water solubility,NaOH has to be ultilized to dissolve realgar in previous basic research,which may lead to chemical changes of realgar,increased the difficulty of mechanism research.In addition,the application of realgar was focused on the treatment of leukemia,but less on its efficacy in solid tumors.In order to solve the above problems.hydrophilic realgar nanoparticles were prepared to increase its dispersibility in water and enhance its bioavailability.The anti-tumor effects and mechanisms of realgar nanoparticles were studied on mouse breast cancer model and chronic myeloid leukemia(CML)cells.Methods:In this study,hydrophilic realgar solid dispersion(nanoparticles)was prepared by hot lelt co-extrusion technology by blending original realgar with an amphiphilic polymer matrix.The size and element distribution of nanoparticles were analyzed by dynamic light scattering,scanning electron microscopy and X-ray energy spectrum.Mouse breast cancer xenograft model was established by injecting mouse breast cancer cells orthotopically into mouse breast fat pad.The therapeutic effects of hydrophilic realgar nanoparticles on breast cancer were studied on mouse breast cancer cell line and breast cancer mouse model.The breast cancer-bearing mice were orally administrated with the nanoparticles,original realgar,polymer matrix and saline and the metastasis and survival of tumors in mice were studied.Reactive Oxygen Species(ROS),superoxide dismutase 2(SOD2),hypoxia-inducible factor 1?(HIF-1?),carbonic anhydrase IX(CAIX),CD31 and inflammatory corpuscle NLRP3 were detected by immunohistochemistry.The co-localization of CD31 and alpha-SMA was detected by immunofluorescence.The transcription levels of BCR-ABL in cells and Hif1a,Ldha and Vegfa in tumor tissues were detected by quantitative Real-Time PCR.The effects and molecular mechalism of hydrophilic realgar nanoparticles on CML were studied by using CML cell lines and bone marrow mononuclear cells(BMMNCs)derived from CML patients.The effects of hydrophilic realgar nanoparticles and original realgar on cell viability were detected by CCK-8.Cell proliferation,differentiation,apoptosis,ROS and cycle distribution were detected by flow cytometry.The erythroid differentiation of CML cells was detected by benzidine staining.Superoxide anions and hydroxyl radicals were detected by electron paramagnetic spectrometer.Autophagosomes were observed by transmission electron microscopy.Western Blot was used to detect the expression of p38,p-p38,ERK1/2,p-ERK1/2,CDC25,p-CDC25,AKT,p-AKT,LC3B,BCR-ABL,CBL,HIF-1?,CD31,CDKNIA proteins.Results:1.Hydrophilic realgar nanoparticles prepared by hot melt co-extrusion technology can be easily dissolved in water,saline or buffer solution to form a yellow colloidal solution.The average hydrated size of realgar particles in solution is 400-700 nm,which is significantly smaller than that of original realgar.The cytotoxicity of realgar nanoparticles in K562 cells,4T1 cells and RAW 264.7 cells is much higher than that of original realgar.2.Hydrophilic realgar nanoparticles could reduce the activity of breast cancer cell 4T1 and macrophage RAW 264.7 and also reduce liver and lung metastasis and prolong the survival of tumor-bearing mice.The mechanistic study showed that hydrophilic realgar nanoparticles could scavenge superoxide anion and hydroxyl radicals in vitro and it could enter the tumor tissues to scavenge ROS and reduce the expression of antioxidant enzyme SOD2 in the tumor tissue.The nanoparticles also down-regulated the expression of HIF-1? by decreasing the expression Hif1a mRNA in the tumor tissues,thereby reducing the downstream transcription of Vegfa and Ldha and the expression of CAIX,and reducing angiogenesis and inflammsome NLRP3;therefore regulating the hypoxic microenvironment and inflammation.3.Hydrophilic realgar nanoparticles could be uptaken by K562 cells,thereby effectively reducing the cell viability,inducing apoptosis,inhibiting the proliferation,and arrestting cell cycle in CML cells.At low concentrations,hydrophilic realgar nanoparticles could induce erythroid differentiation of CML cell lines and BMMNCs derived from CML patients.The mechanistic study showed that hydrophilic realgar nanoparticles induced cell cycle arrest through phosphorylation of ERK1/2 and induce erythroid differentiation through phosphorylation of p3 8 MAPK.The nanoparticles could also induce autophagic degradation of BCR-ABL by scavenging ROS in CML cells.Conclusion:Hydrophilic realgar nanoparticles can effectively scavenge ROS in cells and tissues through their intrinsic reducibility,therefore demonstrating their anti-tumor effects.The nanoparticles inhibited the metastasis effectively in breast cancer-bearing mice and prolonged the survival time of the mice significantly.At the same time,hydrophilic realgar nanoparticles not only achieved higher cytotoxicity,effectively induced K562 cell cycle arrest and apoptosis,but also induced erythroid differentiation in K562 cells and BMMNCs cells derived from CML patients at low concentration.In conclusion,this study provides experimental and theoretical support for the application of hydrophilic realgar nanoparticles in the treatment of breast cancer and CML and the optimization of treatment schemes.