Inhibitory effect of M3 receptor antagonist 4-DAMP on melanoma proliferation of A375 cell line

摘要

Objective: he effect of M3 receptor antagonist 4-DAMP (4-diphenylacetoxy-N-methylpiperidine-methiodide) on the proliferation of melanoma were explored, and the development prospect and significance of M3 receptor antagonist in the field of anti-tumor were discussed. Methods: The viability of tumor cells was detected by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) colorimetry. The optimal concentration of 4-DAMP was screened and the effect of the drug was confirmed by EdU (5-ethynyl-2'-deoxyuridine) fluorescence staining. Adult male BALB/c nude mice were divided into control group, 5-Fu (5-Fluorouracil, 5-Fluorouracil, 20 mg/kg/d) group and 4-DAMP (2 mg/kg/d) group to establishtumor-bearing model in vivo. The tumor volume curve was calculated and plotted. Mice were killed after 21 days of continuous administration. Strip the tumor, take pictures and meatured the weighs, and calculate the inhibition rate. [1]The spleen was weighed and the spleen index was culculated. H & E staining was used to observe the pathological changes of tumor sections. The gene expression of M3R、MIA (Melanoma inhibitory activity, MIA)、SP-1 (Transcription factor Sp1, SP-1) and Lnc-SPRY4-IT1 (SPRY4 Intronic Transcript 1,Lnc-SPRY4-IT1) in A375 cell line was detected by real-time quantitative PCR (quantitative reverse-transcription polymerase chain reaction, qRT-PCR). The protein level of ERK1/2 (extracellular signal-regulated kinase 1/2,ERK1/2) was detected by Western Blot. Results: 4-DAMP could significantly inhibit the activity of A375 cell line (P < 0.01), and EdU staining demonstrated that 4-DAMP inhibited the growth of A375 cell line. The results of animal experiments showed that compared with the control group, the volume and mass of tumor in 5-Fu group and 4-DAMP group were significantly reduced (P < 0.05) and H & E staining showed that the intercellular space became larger, connective tissue increased, and tumor growth was obstructed. Compared with the control group, the tumor inhibition rate of 4-DAMP group was 72.01% (P < 0.01). There was no significant difference in body mass and spleen index. Compared with the control group, the expression of MIA, SP-1 and LncRNA-SPRY4-IT1 was decreased by 4-DAMP or si-M3R transfection. Compared with the control group, the expression of ERK1/2 was decreased by 4-DAMP or si-M3R transfection. Conclusions: These results suggest that M3 receptor antagonist 4-DAMP can inhibit the proliferation of melanoma and reduce the expression of mRNA of MIA, SP-1 and LncRNA-SPRY4-IT1 and the protein level of ERK1/2 in A375 tumor cells.