| Objective:Neuroblastoma(abbreviation,NB)is one of the most common malignant tumors in children.It is the main cause of death in NB patients because of its high degree of malignancy and early metastasis.This high degree of invasion and metastasis has also become a very characteristic phenomenon of NB in the whole malignant tumor diseases.Therefore,exploring the mechanism of NB metastasis,controlling metastasis is the key to determine the prognosis,and it is also the most challenging problem in the treatment of NB.At present,high-risk NB patients with high degree of malignancy and early metastasis are often treated with super-high-dose combined chemotherapy.Although it is effective at the beginning,the toxicity and side-effects of chemical synthetic drugs are high.It is easy to recur,and the effect is not good.However,natural compounds have the characteristics of targeted killing of tumor cells,which is the focus of anti-tumor research at present.Therefore,it is of great significance to search for natural drugs against NB transfer.Our research group has been studying isatin for many years.In 1999,we first reported at the international conference that isatin has an anti-oxygen free radical effect,which can protect normal nerve cells and antagonize the death of nerve cells caused by environmental toxins.Isatin was found to have an anti-tumor effect.Isatin,which can protect normal cells through anti-tumor free radicals,is the property of most anti-tumor agents at present.Isatin has the advantages of low molecular weight,oral administration,targeting inhibition of tumor cell proliferation,avoiding damage to normal cells,and low toxicity and side effects.It is a natural small molecular compound drug of great value for development.In this study,NB metastatic tumor cell lines and visualized animal models were used to clarify the anti-NB metastasis effect of isatin and to explore its possible molecular mechanism.The aim of this study was to elucidate the anti-NB metastasis effect of isatin and its molecular mechanism in order to find out the potential targets of NB metastasis therapy.Methods:In vitro culture of neuroblastoma cell line SH-SY5 Y,the effect of different concentrations of isatin on proliferation activity of NB cells was detected by CCK8 assay.Soft Agar colony forming assay was used to detect the effect of isatin on colony formation ability of SH-SY5 Y cells.The migration ability of SH-SY5 Y cells treated with isatin(0,50,100,200 μmol/L)was determined by scratch assay.Changes in invasion and motility of SH-SY5 Y cells treated with different concentrations of isatin(0,50,100,200 μmol/L)were determined by transwell assay.The effect of different concentrations of isatin on apoptosis,cell cycle and mitochondrial transmembrane potential of SH-SY5 Y cells were detected by Flow cytometry(FCM).Furthermore,fluorescence staining,RT-PCR and Westernblot were used to observe the effects of isatin at different concentrations on the proliferation,apoptosis,invasion and migration of SH-SY5 Y cells.LSD1 expression in tumor tissues of 45 patients with neuroblastoma was detected by Immunohistochemistry(IHC).Lentivirus vector pLenti-LUC was co-transfected into 293 T cells to prepare lentivirus.Infection this lentivirus to neuroblastoma SH-SY5 Y cells,screening them with achromycin quartic to express strong signals from cells,continue to expand in vitro culture,the luciferase marked of SH-SY5 Y cells were injected into the left ventricular of nude mice to form model of metastatic neuroblastoma.In vivo fluorescence signal was monitored by in vivo imager.The inhibition of isatin on the metastatic tumor model in nude mice was observed,and the metastasis of various major organs in nude mice treated with isatin was detected,and the anti-tumor activity and mechanism of isatin in vitro and in vivo were also discussed.Results:1.The effects of isatin(0,50,100,200 μmol/L)on cell proliferation,migration,invasion,apoptosis,mitochondrial transmembrane potential and cell cycle of NB cell line SH-SY5 Y,were examined after 48 h treatment with isatin(0,50,100,200 μmol/L).The results of CCK8 assay showed that isatin could significantly inhibit the proliferation of SH-SY5 Y cells.In soft Agar colony formation test,SH-SY5 Y cells were cultured for 14 days after treated with isatin for 48 h,took photos to observe the number of colony formation of SH-SY5 Y cells.Isatin could significantly inhibit the colony formation of SH-SY5 Y cells,which further confirmed the inhibitory effect of isatin on cell proliferation.The results of scratch test showed that the migration ability of the cells treated with isatin was lower than that of the control group(P < 0.05),and showed a concentration-dependent manner.Compared with the control group,the invasiveness of the cells decreased(P < 0.01),and the results also showed a concentration-dependent manner in transwell assay.200μmol/L isatin was observed under fluorescence microscope.Typical morphological changes of apoptosis were observed in cells treated with isatin,such as nuclear condensation andnucleus.After treatment with isatin,the apoptosis rate of SH-SY5 Y cells increased with the increase of the concentration of isatin,and the number of apoptotic bodies increased with the increase of the concentration of isatin.By FCM,the apoptosis rate of SH-SY5 Y cells increased with the addition of FITC-Annexin V/PI to the treatment of isatin.FCM assay was used to detect the cell cycle.The results showed that after treated with 50,100,200 μmol/L isatin for 48 h,the number of SH-SY5 Y cells in G1 phase increased significantly and showed a significant G1 phase arrest.SH-SY5 Y cells were treated with different concentrations of isatin for 48 h.The mitochondrial membrane potential was detected by FCM after Rhodamine 123 staining.The results showed that the mitochondrial membrane potential decreased significantly in a concentration-dependent manner after treatment with different concentrations of isatin.2.Real-time quantitative(Real-time)PCR,Western blot(Westernblot),immunofluorescence and immunoprecipitation were used to detect the changes of signal pathway in SH-SY5 Y cells treated with isatin.The results showed that isatin could inhibit the proliferation of SH-SY5 Y cells.The ability of invasion and migration may,on the one hand,induce apoptosis by decreasing the mitochondrial membrane potential,and on the other hand,isatin can act on the MAOA,of monoamine oxidase on the outer membrane of mitochondria by inhibiting the cell metastasis-related reactive oxygen species(ROS),Hypoxia inducing factor HIF-1α,chemokine receptor CXCR4,Matrix Metalloproteinase(MMP2,MMP9),and Vascular Endothelial Growth Factor(VEGF),thus inhibiting the survival,proliferation,angiogenesis,invasion and metastasis of tumor cells.Moreover,the small molecular weight of isatin may directly inhibit the activity of monoamine oxidase lysine-specific histone demethylase(LSD1),thus altering the methylation of the lysine k4 site of histone H3 to make the H3K4 single.The level of double methylation increased,which changed the regulation of gene expression.And isatin suppressed the expression of LSD1 could up-regulate the expression of p53 gene and further activate the downstream gene p21 protein.P21 protein could inhibit cyclin-dependent kinase complex and arrest cell mitosis in G1 phase.On the other hand,p53 can promote the apoptosis of cancer cells by up-regulating the expression of Bax and down-regulating the expression of Bcl-2 and MDM2.In addition,because isatin inhibited the activity of LSD1,which reduced the binding of pNF-κb to LSD1,thus inhibited the activity of ERK,and also affected the activation of ERK by TGF β1.Isatin inhibited the expression of TGF β1 which inhibited the EMT process of tumor invasion and metastasis.Therefore,we infer that isatin may be able to inhibit the metastasis of neuroblastoma cells through multiple pathways.3.Immunohistochemical method was used to detect the expression of LSD1 in 45 cases of neuroblastoma,44 cases were positive and 1 case was negative.The positive expression rate was 97.78%.LSD1 was highly expressed in tumor tissue of NB patients.4.The metastasis model of neuroblastoma in nude mice was established by injecting SH-SY5 Y cells labeled with Luc into the left ventricle.The nude mice were randomly divided into four groups: model group,positive drug cyclophosphamide(CTX 40 mg/kg)group,isatin treatment group(isatin 200 mg/kg),positive drug and isatin combination group(CTX 20 mg/kg and isatin 200 mg/kg).To observe the formation and treatment of metastatic foci in nude mice by means of living imaging apparatus of small animals every week.After four weeks of administration,the results showed that compared with the model group,there was no significant difference in the weight change of nude mice between the model group,isatin treatment group,the positive drug CTX group,and the combination group of isatin and CTX(P > 0.05).Compared with the model group,other three groups could reduce the fluorescence intensity of metastasis in nude mice(P < 0.01).Compared with model group,the fluorescence signal intensity of in vitro imaging of bone metastases in nude mice was decreased in three treatment groups(P < 0.01),and the intensity of in vitro imaging of main organs in nude mice was decreased in all treatment groups(P < 0.01),and the difference was significant(P < 0.01).The results of detection of tumor metastasis-related factor VEGF,MMP2,MMP9 in serum of nude mice in each group showed that the content of each factor in the model group was significantly higher than that in the other three groups(P < 0.05,P <0.01),and the concentration of VEGF in the model group was significantly higher than that in the other three groups.Compared with the model group,the content of ISA group and CTX + ISA combined group was lower than that of the model group(P < 0.05).The concentration of MMP2,MMP9 in the other groups was lower than that in the model group(P < 0.05,P < 0.01).The levels of SOD(superoxide dismutase)and GSH-PX(glutathione peroxidase),MDA(malondialdehyde)in serum of nude mice in each group were measured.The results showed that the activity of SOD,GSH-PX in CTX group was significantly lower than that in the other three groups,and the activity of SOD,GSH-PX in CTX group was significantly lower than that in other three groups.The difference was statistically significant(P < 0.05).The concentration of MDA was significantly higher than that of the other three groups(P < 0.05,P < 0.01).The results showed that the contents of creatinine and urea nitrogen in the CTX group were significantly higher than those in the other three groups(P <0.05,P < 0.01).The results showed that the serum total bilirubin(Tibl),GGT(γ-GT),AKP,ALT,AST of serum in CTX group was was significantly higher than that of the other threegroups(P < 0.05,P < 0.01).There was no significant difference in serum calcium,potassium and sodium electrolytes among the three groups(P > 0.05).Conclusion:1.Isatin can inhibit the proliferation,invasion and migration of SH-SY5 Y cells,induce apoptosis and cause cell cycle arrest.2.The results of cell-level molecular mechanism experiment showed that isatin may produce anti-metastasis effect through multiple pathways.First,it may be through MAOA/HIF/CXCR4 signaling pathway,and inhibit the activation of VEGFR1,and reduce the level of ROS.Inhibiting tumor angiogenesis;Second,it is possible to increase the expression of p53 and promote cell apoptosis through LSD1/p53-mediated apoptosis pathway.3.In vivo,Luc-labeled SH-SY5 Y metastatic tumor model in nude mice was successfully prepared by intracardiac injection of tumor cells.Isatin can reduce the metastasis of NB cells in nude mice.The combination of isatin and cyclophosphamide can enhance the anti-metastasis effect of cyclophosphamide,and reduce the toxic side effects of liver and kidney injury caused by cyclophosphamide,and produce synergistic and attenuated side effects. |
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