Study On The Anti-melanoma Effect And Mechanisms Of Hydroxyapatite Nanoparticles And Its Hydrogel

Malignant melanoma(MM)is a highly malignant tumor originating from melanocytes,which is prone to recurrence and metastasis,and is the highest mortality in skin tumors.Its pathogenesis is unknown.Recent studies have shown that the occurrence,progression and metastasis of MM are closely related to a variety of mutant genes and the activation of downstream signal pathways.The main treatments of MM are surgical resection combined with radiotherapy,chemotherapy,targeted therapy and immunotherapy.However,the therapeutic effect is not ideal,and the patients are prone to local recurrence and metastasis after treatments.Therefore,it is necessary to find new drugs or new materials that can reduce local recurrence and do not have obvious side effects for MM treatment.Hydroxyapatite nanoparticle(n HAP)is a natural,nano-sized calcium apatite compound with good biocompatibility,which is widely used in the field of drug carriers and bone tissue repair.In recent years,it has been reported that n HAP has inhibitory effect on different kinds of tumor cells,while it has almost no effect on normal cells.In our previous study,we screened the needle-like n HAP(the average particle size is 50nm)particles with the greatest inhibitory effect on the proliferation of MM cells,and confirmed that n HAP could selectively inhibit the proliferation,invasion in vivo and in vitro.It could also increase the level of reactive oxygen species(ROS)in melanoma cells.According to the transcriptome results,it was inferred that the anti-MM effect of n HAP particles may be related to the mitochondrial-dependent apoptosis pathway.Therefore,in this study,human melanoma cell line SK-MEL-28 and A375,and human normal skin immortalized keratinocyte Ha Ca T were used as subjects to verify the results of previous transcriptome speculation firstly,and then to explore the possible protein changes when n HAP inhibits MM from the protein level,and to further study the possible mechanisms of n HAP.Finally,we prepared the hydrogel loaded with needle-like n HAP particles,established the MM implantation material model in nude mice,observed and compared the efficacy and safety between n HAP particles and n HAP-loaded hydrogel in inhibiting MM,so as to provide a theoretical basis for the preclinical application of n HAP.Objective:1.To explore whether n HAP particles has the effect on apoptosis of melanoma cells and its possible mechanisms by in vitro study.2.To explore the inhibitory effect of n HAP particles and n HAP-loaded hydrogel on melanoma,and to investigate whether n HAP-loaded hydrogel has biological safety and compatibility by in vivo study.Materials and Methods:1.Exploring the apoptosis effect on melanoma of n HAP particles in vitro:different concentrations group(60,120,240,480 μg/m L)of n HAP particles and blank control group(no adding n HAP particles).(1)To observe the effect of n HAP particles on apoptosis of SK-MEL-28,A375 and Ha Ca T cells by Hochest 33258 and Annexin V-FITC/PI.(2)To observe the changes of intracellular organelles after n HAP particles entering in SK-MEL-28/A375/Ha Ca T cells by transmission electricity microscopic.(3)To evaluate the change of intracellular redox level of SK-MEL-28/A375/Ha Ca T cells by measuring the redox index intracellular glutathione(GSH),catalase(CAT),superoxide dismutase(SOD)and malondialdehyde(MDA).(4)To observe the effect of n HAP particles on apoptosis-related proteins(Bax,Bad,cyt-c,Bcl2,p53)by western blot(WB).2.Exploring the possible anti-melanoma mechanisms of n HAP particles at protein level:(1)the proteins of human melanoma cells SK-MEL-28 treated with or without n HAP(240 μg/m L)for 24 hours were sequenced by Tandem Mass(TMT)technique,and the differential proteins(DEPs)obtained were analyzed by GO(gene ontology)and KEGG(kyoto encyclopedia of Genes and genomes)analysis;(2)The significant differential proteins by TMT were identified by WB to determine the possible mechanisms of n HAP anti-melanoma.3.Evaluating n HAP particles and n HAP-loaded hydrogels in inhibiting melanoma in vivo:(1)The hydrogels based on polymer materials such as sodium alginate(Alg)and chitosan(CS)were prepared,then needle-like n HAP(the average particle size is 50nm)was loaded into the hydrogel system to prepare composite hydrogels with slow release of nanoparticles.(2)Melanoma animal model was established by subcutaneous injection of SK-MEL-28 and A375 cells into the back of Balb/c nude mice.(3)when the length and short diameter of the tumor were approximate 3mm,each model was randomly divided into 4 groups and subcutaneously implanted with the same dose of n HAP particles,Alg-CS-n HAP hydrogels,Alg-CS hydrogels and PBS.(4)the general condition and tumor volumes were observed and recorded by Vernier caliper every other day.(5)At the end of the experiment,28 days after implantation,the weight and volume of tumor were measured;Tunnel was used to detect apoptosis;the morphology of melanoma was observed by haematine-eosin staining(HE)and the expression of Cleaved-Caspase 3,Bax,cyt c,Bcl-2,p53 in tumor tissues were detected by immunohistochemistry(IHC).(6)The organs of mice in each group were stripped,and the morphology of heart,liver,spleen,kidney and lung,were observed by visual observation and light microscopy to evaluate the biocompatibility and biological safety.(7)At the end of the experiment,eyeball blood samples were taken to detect blood routine,liver and kidney function,blood lipids,and blood calcium to evaluate the safety of n HAP particles,Alg-CS-n HAP,Alg-CS hydrogel particles and hydrogel in vivo.Results:Part 1 Effects of hydroxyapatite nanoparticle on apoptosis of melanoma cell line SK-MEL-28 and A375(1)The results of Hoechst33258 staining in SK-MEL-28,A375 and Ha Ca T cells treated with different concentrations of n HAP particles were compared.In SK-MEL-28 and A375 cells,the cells treated with n HAP particles had different amounts of bright blue fluorescence,and strong bright blue phenomenon of micronucleus.Nuclear fragmentation could be seen in the n HAP group of 240 μ g /ml and 480 μ g /ml,which were the characteristics of apoptotic cells.However,no similar phenomenon was observed in each group of Ha Ca T cells.Annexin V-FITC/PI detection showed that n HAP could promote the apoptosis of A375 cells in a concentration-dependent manner,while n HAP of 240,480 μg/m L could promoted the apoptosis of SK-MEL-28 as well(P < 0.001),but it had little effect on Ha Ca T cells(P > 0.05).(2)After SK-MEL-28,A375 and Ha Ca T cells were treated with different concentrations of n HAP for 24 hours,both mitochondria and endoplasmic reticulum swelled were observed in melanoma cells in 240,480 μg/m L of n HAP,but there was no related change to Ha Ca T cells by transmission electron microscopy.(3)After SK-MEL-28 and A375 cells were treated with different concentrations of n HAP particles for 48 hours,the levels of GSH,SOD and CAT decreased,but MDA increased.Consequently,the redox balance of MM cells was destroyed,but Ha Ca T had no similar change.(4)After the cells were treated with different concentrations of n HAP for 48 hours,comparing with the control group,the expression of anti-apoptotic protein Bcl-2 decreased,while the expression of pro-apoptotic proteins Bax,Bad,cyt c and p53 increased.Similar expression of these proteins were not detected in Ha Ca T cells.These results suggested that n HAP particles selectively promoted the apoptosis in MM by activation of mitochondrial-related apoptotic pathways.Part 2 The possible mechanisms of hydroxyapatite nanoparticle on the anti-melanoma effect by proteomics technology(1)A total of 130 differentially expressed proteins,including 62 up-regulated proteins and 68 down-regulated proteins,were screened.(2)According to the bioinformatics analysis of the selected differential proteins,GO analysis was mainly concentrated in five aspects: calcium binding,protein hydrolysis,regulation of cell growth,Wnt signal pathway and cell adhesion.KEGG analysis was mainly concentrated in Wnt,protease hydrolysis of tumor,p53 signal pathway,oxidative phosphorylation signal pathways.According to the above results,six target differential proteins of THBS1,APOA1,DKK1,WNT5 a,IGFBP3 and IGFBP5 were screened.(3)The results of WB detection showed that compared with the blank control group,Wnt pathway related proteins WNT5 a and DKK1(P < 0.001)were significantly higher.The expression of p53 pathway related proteins THBS1 and IGFBP3 were increased significantly(P< 0.001).The expression of protein APOA1 related to protease hydrolysis was significantly increased(P < 0.001).These results were consistent with those of TMT sequencing.It showed that the inhibition of n HAP particles on MM may be related to THBS1,APOA1,DKK1,WNT5 a,IGFBP3 proteins.Part 3 Study on the anti-melanoma effect and mechanisms of n HAP-loaded hydrogel in mice(1)The animal model of melanoma plus implantation of materials in Balb/c nude mice was established successfully.(2)Visual observation: Both the n HAP particles and Alg-CS-n HAP hydrogels could inhibit the growth of SK-MEL-28 and A375 tumor in vivo,while Alg-CS had no inhibitory effect on tumor.At the end of the experiment,there was no significant difference in tumor inhibition between the n HAP particles group and Alg-CS-n HAP hydrogels group.But we found that the action time of Alg-CS-n HAP hydrogels was later than that of n HAP group,which may be related to the slow release of nanoparticles from the hydrogel system.(3)Tunnel was positive in n HAP particles and hydrogels group(P<0.001).n HAP could up-regulate the expression of apoptosis protein of Cleaved-Caspase-3,cyt c,Bax,p53 and down-regulate Bcl-2(P<0.001).These results indicated that n HAP inhibited MM and the possible mechanisms were that it may activate the mitochondrial-dependent apoptotic pathway and promote the apoptosis of MM by in vivo study.(4)The implanted materials did not cause inflammatory reaction to surrounding skins or main viscera,indicating that n HAP had no obvious side effects on local tissue and system.(5)The implanted materials did not affect the blood routine,liver and kidney function,blood lipid and calcium metabolism of Balb/c nude mice,which further indicated that n HAP had no obvious side effects on the system.Conclusions:1.This study proved that n HAP particles could destroy the redox balance of MM cells,and activate the apoptosis pathway related to mitochondria in vitro.Its mechanisms may be related to the regulation of Wnt,p53,oxidative phosphorylation signal pathway,which in turn affects THBS1,APOA1,DKK1,WNT5 a and IGFBP3 proteins.Therefore,it is speculated that the mechanisms of n HAP inhibiting MM may be related to multiple pathways and several targeted proteins.2.n HAP may activate mitochondrial-related apoptosis pathway.The hydrogel loaded with n HAP has the function of slow release of nanoparticles,which may be the reason of the later time of inhibiting tumor.3.The n HAP-loaded hydrogels has good biocompatibility and biosafety.Therefore,it is expected to be a new biological material for preventing local recurrence of after treatment of MM in the future.