Antitumor Effect And Mechanism Of Human Fetal Dermal Mesenchymal Stem Cells On Melanoma Cells

Research backgroundMalignant melanoma(malignant melanoma)is a malignant tumor caused by high malignant transformation of melanocytes.Histology and embryology come from neural crest cells,most of which occur in the skin.The incidence of MM is the third highest in all skin malignant tumors.Compared with other malignant tumors,the incidence of malignant melanoma is not high,accounting for about 1%to 3%of all malignant tumors,but the incidence of melanoma has increased year by year in recent years.Melanoma has a high rate of metastasis,blood and lymphatic metastasis occurs in the early stage,and the mortality rate of patients is high.Authoritative data report:the five-year survival rate of patients with malignant melanoma metastasis is less than 10%.The risk factors of malignant melanoma include the following aspects:gene sensitivity,excessive ultraviolet and environmental stimulation,history of nevus,etc.,but the specific etiology is still unclear.At present,the main treatment of melanoma is surgical local resection,regional lymph node dissection,postoperative adjuvant radiotherapy and conventional chemotherapy.At present,the research and development and application of anti-tumor adjuvant therapy(such as targeted drugs and biological immunity,etc.)have also made some progress,but most of the drugs have many side effects and the curative effect is very limited.Therefore,we need to re-understand this disease,explore the pathogenesis of melanoma,clarify its diagnosis,staging and classification,and seek a more effective treatment to fundamentally improve the therapeutic effect of malignant melanoma.Mesenchymal stem cells(MSCs)is a kind of pluripotent stem cells derived from matrix.MSCs has a variety of tissue sources,can be found in bone marrow,synovium,adipose tissue,periodontal ligament and skin,and has the characteristics of easy separation and amplification,low immunogenicity,and MSCs has multi-directional differentiation potential and immunomodulatory ability.It has a broad application prospect in the field of regenerative medicine and tissue engineering.It can promote the repair and regeneration of cardiovascular,nervous system and urinary system,and can be used in the treatment of glaucoma,diabetes,myocardial infarction and other diseases.In addition,the interaction between MSCs and tumor has also attracted wide attention of researchers.A large number of literatures have confirmed that MSCs has the ability to homing to tumor tissues and can inhibit the progress of tumor tissues.In the previous study,our team found a new repository of MSCs-fetal dermis,and successfully isolated human fetal dermal mesenchymal stem cells(FDMSCs).Our previous study found that the paracrine factor of FDMSCs can inhibit the growth of keloid,which provides a new treatment strategy for the treatment of keloid.At present,whether FDMSCs can be used in the treatment of malignant melanoma has not been reported in the literature.Research purposeIn vitro,we studied the effect of FDMSCs conditioned medium(conditioned media from FDMSCs,CM-FDMSC)on the biological activity of A375 melanoma cells and its possible mechanism.In vivo,we studied the effect of CM-FDMSC on A375 melanoma cell animal model.Through two aspects,we explore the feasibility of FDMSCs related therapy in the treatment of malignant melanoma and provide an experimental and theoretical basis for the future use of stem cell therapy in the treatment of clinical melanoma patients.The details are as follows:1.The effect of CM-FDMSC on the activity and proliferation of A3 75 melanoma cells was measured in vitro.2.To evaluate whether CM-FDMSC induces apoptosis of A375 melanoma cells in a dose-and time-dependent manner.3.The apoptosis of A3 75 melanoma cells induced by CM-FDMSC was determined by Annexin V-FITC/PI double staining assay.4.The apoptosis of A3 75 melanoma cells induced by CM-FDMSC was determined by TUNEL assay.5.Whether CM-FDMSC inhibited the migration of A375 melanoma cells was verified by scratch healing test.6.The effect of CM-FDMSC on the invasiveness of A375 melanoma cells was determined by Transwell invasion test.7.After the intervention of CM-FDMSC in A375 melanoma cells,the changes of PI3K/AKT pathway,MAPK pathway and mitochondrial-mediated internal apoptosis pathway in A375 melanoma cells were observed.8.To establish the animal model of A375 melanoma cells and evaluate the effect of CM-FDMSC on the tumorigenesis of A375 melanoma cells.Research methods1.Extraction and identification of FDMSCs,further preparation of FDMSCs conditioned medium(CM-FDMSC)by FDMSCs,different concentrations of conditioned medium were made by centrifugal filtration device,and the blank control group was set up.The concentration gradient group was 0.5 x CM conditioned medium(low concentration group),1 × CM conditioned medium(medium concentration group)and 2 x CM conditioned medium(high concentration group).A375 melanoma cell line was obtained from American Type Culture Collection(ATCC)in the United States,and verified by short tandem repeat(STR)analysis.2.The effects of different concentrations of CM-FDMSC on A375 melanoma cells were studied by CCK-8 assay.Melanoma cells planted in a 96-well plate were treated with different concentrations of conditioned medium.Two days later,CCK-8 solution was added and incubated for three hours to determine the changes of absorbance.3.EdU binding assay was used to verify the effect of different concentrations of conditioned medium on the proliferation of A375 melanoma cells.A375 melanoma cells treated with different concentrations of CM-FDMSC were implanted in a six-well plate,and EdU test solution was added for two hours.Then all nuclei were stained with Hoechst 33342,and the proportion of EdU positive nuclei to Hoechst 33342 stained nuclei was calculated.4.The apoptosis of A375 melanoma cells treated with different concentrations of CM-FDMSC was analyzed by flow cytometry.After A375 melanoma cells were treated with different concentrations of CM-FDMSC for two days,AnnexinV-FITC and PI were added to detect the number of apoptotic cells.At the same time,0,12,24,48 and 72 h time points were designed to detect whether apoptosis was time-dependent.5.TUNEL assay was used to detect the effect of different concentrations of CM-FDMSC on the apoptosis rate of A3 75 melanoma cells.After A3 75 melanoma cells were treated with different concentrations of CM-FDMSC for 2 days,A375 melanoma cells were incubated with TUNEL detection solution for 1 hour,then stained with DAPI,and the proportion of apoptotic cells was calculated after taking pictures by confocal microscope.6.The effects of different concentrations of CM-FDMSC on the migration of A375 melanoma cells were detected by the scratch assay.A375 melanoma cells were placed in a 6cm petri dish.After monolayer cells were formed,monolayer cells were scratched.Two days later,the effects of different concentrations of CM-FDMSC on the migration ability of A375 melanoma cells were observed.7.Transwell invasion assay was used to detect the regulation of different concentrations of CM-FDMSC on the invasion of A375 melanoma cells.A375 melanoma cells treated with different concentrations of CM-FDMSC were added to the upper chamber and the lower chamber was added to the complete medium containing serum.After 14 hours,the number of A375 melanoma cells invading the lower chamber was observed.8.The expression of key signaling pathway proteins and their phosphorylated proteins in PI3K/AKT pathway,MAPK pathway and mitochondrial-mediated internal apoptosis pathway of A375 melanoma cells treated with different concentrations of CM-FDMSC were detected by Western blotting technique.The changes of related genes in the PI3K/AKT signaling pathway were evaluated by quantitative real-time fluorescence PCR(qRT-PCR).9.A375 melanoma cells treated with different concentrations of CM-FDMSC were injected subcutaneously into the right axillary skin of 4-6-week-old nude mice.Five days later,the changes of tumor volume and weight were measured every three days,and the nude mice were killed 17 days later.The tumor tissues of different groups of tumor-bearing nude mice were removed,weighed and histopathologically examined.Research result1.We can extract FDMSCs successfully.The extracted cells accorded with the morphological characteristics of mesenchymal stem cells under an inverted microscope and detected by flow cytometry.The extraction of cell surface antigens CD44,CD90 and CD 105 met the international standards for mesenchymal stem cells.It does not express the common surface antigen markers of hematopoietic stem cells(excluding)and has the ability of osteogenesis,chondrogenesis and adipogenic differentiation.Compared with DSMZ database cells,our cells accorded with the characteristics of A375,and the corresponding cell number CRL-1619,completed the identification of A375 melanoma cells.2.CM-FDMSC can inhibit the proliferation of A375 melanoma cells.In addition,we found that with the increase of CM-FDMSC concentration,the proliferation rate of A375 melanoma cells decreased in a dose-dependent manner.3.Through EdU binding assay of A375 melanoma cells,we observed the microscopic changes of A375 melanoma cells under a microscope.It was found that the proportion of EdU positive nuclei in different concentration groups was about the same,and there was no significant statistical significance.4.By analyzing the distribution of AnnexinV-FITC/PI double staining by flow cytometry,we observed that A375 melanoma cells treated with different concentrations of CM-FDMSC could produce apoptosis in a dose-dependent manner.At different time points,we found that CM-FDMSC induced apoptosis of A375 melanoma cells in a time-dependent manner(with the prolongation of CM-FDMSC treatment time,the apoptosis rate of A375 melanoma cells increased).5.Using confocal microscopy,we observed that the proportion of TUNEL staining cells in DAPI staining increased with the increase of CM-FDMSC concentration,and there was statistical significance between different concentration treatment groups.It is dose-dependent.6.Through the scratch test,we found that the migration ability of A375 melanoma cells treated with different concentrations of CM-FDMSC was inhibited.The difference of healing ratio in different concentration treatment groups was statistically significant.7.Through the Transwell invasion test,we also found that different concentrations of CM-FDMSC inhibited the invasion of A375 melanoma cells.A375 melanoma cells treated with different concentrations of CM-FDMSC were added to the upper chamber and the lower chamber was added to the complete medium containing serum.After 14 hours,the number of invasive cells of A375 melanoma cells was observed.Compared with the control group,we found that the number of cells on the lower surface of the ependyma on Transwell decreased with the increase of CM-FDMSC concentration.The experimental results showed that there was a significant difference between different concentration groups.8.Using Western blotting technique,we detected the changes of the protein expression level of the three pathways.After CM-FDMSC treatment,the phosphorylation of PI3K,AKT,and ERK protein decreased,the expression of BAX protein was up-regulated,and the expression of BCL-2 protein was down-regulated.The total PI3K,AKT and ERK proteins did not change much,in which the ratio of BCL-2/BAX became smaller,and the overall trend developed towards the direction of promoting apoptosis.There were statistical differences among different concentration treatment groups.At the same time,we detected the changes of PI3K and AKT gene levels in the PI3K/AKT signaling pathway by real-time fluorescence quantitative PCR(qRT-PCR).Both methods showed that both PI3K/AKT and MAPK signaling pathways were inhibited in A375 melanoma cells.9.By examining the tumor volume and weight of nude mice,we found that with the increase of CM-FDMSC concentration,the tumor volume and weight of different concentration treatment groups showed a downward trend.Histologically,we found that the number and density of A375 melanoma cells decreased gradually with the increase of CM-FDMSC concentration.With the increase of CM-FDMSC concentration,the connective tissue of tumor tissue in tumor-bearing nude mice became rich,and the thickness of the tumor capsule increased gradually.Research conclusionCCK-8 assay,EdU binding test,AnnexinV-FITC/PI double staining,TUNEL test,scratch test,Transwell invasion test,Western blotting test and animal tumor formation test were used to detect the anti-tumor effect of CM-FDMSC on A375 melanoma cells.1.CM-FDMSC inhibited the tumor activity of A3 75 melanoma cells in a dose-dependent manner in vitro,showing a decrease in cell activity,migration and invasion.2.After A375 melanoma cells were treated with CM-FDMSC,the apoptosis of A375 melanoma cells was dose-and time-dependent.3.After A375 melanoma cells were treated with CM-FDMSC,CM-FDMSC could promote the late apoptosis of A375 melanoma cells.Further studies have found that it may play an anti-tumor role by regulating PI3K/AKT,MAPK and mitochondria-mediated apoptosis signal transduction pathway.4.CM-FDMSC also inhibited the formation of A3 75 melanoma cells in nude mice in animal experiments.CM-FDMSC could inhibit the tumorigenesis of A3 75 melanoma cells.CM-FDMSC changed the structure and composition of A375 melanoma cells in nude mice.Research significanceFDMSCs is a novel mesenchymal stem cell,which has not been reported for the treatment of A375 melanoma at home and abroad.This study confirmed for the first time that CM-FDMSC could inhibit the proliferation and induce apoptosis of A375 melanoma cells,and CM-FDMSC could inhibit the migration and invasion of A375 melanoma cells.In order to explore the deep causes of the anti-tumor effect of CM-FDMSC,we observed the effects of CM-FDMSC on the pathway proteins and genes of A375 melanoma cells from three signal transduction pathways.The three signal transduction pathways are PI3K/AKT,MAPK and mitochondrial mediated apoptosis signal transduction pathways.The discovery of these pathways could help us understand the pathogenesis of A375 melanoma cells.It is expected to further improve the therapeutic effect of A375 melanoma cells.Importantly,we found that CM-FDMSC also inhibited the formation of A375 tumor-bearing nude mice,and it is hoped that relevant pre-clinical trials will be conducted in the future,so as to bring our findings to clinical practice and improve the efficacy of melanoma patients.