Effects Of Natural Indole Compounds On The Proliferation And Metastasis Of Neuroblastoma By Inhibiting LSD1

Objective:Neuroblastoma（NB）is a common malignant solid tumor in children with rapid growth and easy metastasis.Super-large dose combination is the main treatment method for NB,but most chemotherapy drugs are cytotoxic drugs,so it is particularly necessary to find natural compounds that target to kill tumors and have small toxic and side effects.With the deepening of research,indoles have played a variety of roles in organisms.Studies have shown that indole-2,3-dione（Isatin）has an inhibitory effect on monoamine oxidase（MAO）.Lysine-specific demethylase 1（LSD1）,a member of the MAO family,is a popular target for clinical cancer therapy.LSD1 is highly expressed in clinical NB cases.It is of great significance to develop natural indole inhibitors of LSD1 for the treatment of disease.Indole-3-carbinol（I3C）a natural indole small molecule anti-tumor compound commonly found in cruciferous plants,ISA and I3C have the advantages of low toxic and side effects and oral administration,so they are natural small molecule compounds with great development value.In this study,SH-SY5Y cell line was used to clarify the effects of ISA and I3C on the proliferation and metastasis of neuroblastoma and to explore the molecular mechanism of their effects.Methods:（1）CCK8 assay was used to detect the proliferation activity and lethal 50%concentration(IC₅₀)of SH-SY5Y cells treated by ISA and I3C.（2）Colony forming ability of SH-SY5Y cells treated with ISA and I3C was detected by plate cloning.（3）The effect of ISA and I3C on SH-SY5Y cell migration was detected by scratch healing assay.（4）DAPI staining was used to detect the apoptotic bodies after drug treatment.（5）The cell cycle distribution was detected by flow cytometry after PI staining.（6）The m RNA relative expression levels of LSD1,DDIT4 and mTOR in SH-SY5Y cells treated with ISA and I3C were detected by RT-q PCR.（7）The protein relative expression changes of LSD1,DDIT4,mTOR,P70S6K and LSD1 enzyme substrate H3K4me1/2 protein were detected by Western Blot.Results:（1）After 48 h and 72 h treatment with ISA and I3C,SH-SY5Y cell viability decreased with increasing time and concentration（P<0.01）and the IC₅₀values of ISA and I3C on SH-SY5Y cells were 306μmol/L and 121μmol/L.The final concentration of ISA was determined as 0,50,100 and 200μmol/L.The final concentration of I3C was 0,30,60,90μmol/L.The optimal time of action was 48 h.（2）Both ISA and I3C could significantly inhibit SH-SY5Y cell clone number formation（P<0.01）,further confirming the inhibitory effect of the two drugs on cell proliferation.（3）After treatment with drugs for 48 h,the rate of scratch healing in the dosed group was significantly lower than that in the undosed group（P<0.01）.（4）After the treatment of ISA and I3C,the apoptosis rate of the undosed group was 8.18%,8.48%respectively,the apoptosis rate of ISA 200μmol/L and I3C 90μmol/L group was 40.12%and 38.29%,respectively.（5）The cells were significantly arrested in G1 phase by flow cytometry with PI staining and the difference was statistically significant（P<0.01）.（6）With the increase of drug concentration,the m RNA relative expression levels of LSD1 and mTOR were decreased compared with those in the non-dosing group（P<0.01）,the m RNA relative expression level of downstream target DDIT4 was up-regulated（P<0.01）.（7）The relative expression levels of LSD1 and mTOR proteins were decreased compared with those in the untreated group（P<0.01）,the relative expression levels of DDIT4,H3K4me1/2 and P70S6K proteins were increased（P<0.05）.Conclusion:（1）The cell viability,cloning-forming ability and migration ability of SH-SY5Y cells were significantly decreased after being treated by ISA and I3C.In addition,both drugs could induce the apoptosis of SH-SY5Y cells and block the G1 phase.（2）By RT-q PCR and Western Blot,the effect of ISA and I3C on the proliferation and migration of neuroblastoma may be mediated by LSD1-DDIT4/mTOR pathway.（3）In addition,when achieving the same effect,the concentration of I3C was lower than ISA.In view of the high expression of LSD1 in a variety of tumors,the development and research of LSD1 inhibitors is an unmet need in clinical practice.This study provides a new idea for the selection of drugs against neuroblastoma.