4-DAMP抑制A375黑色素瘤增殖作用及机制研究

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

目的:研究毒蕈碱型M3受体拮抗剂(4-diphenylacetoxy-N-methylpiperidine-methiodide,4-DAMP)对黑色素瘤增殖的抑制作用,从而探讨M3受体拮抗剂在抗肿瘤领域的发展前景及意义。方法:利用MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide]检测肿瘤细胞活力;EdU荧光染色观察细胞增殖情况。成年雄性BALB/c裸小鼠分为对照组、5-氟尿嘧啶(5-Fluorouracil, 5-Fu)组(20 mg·kg-1·d-1)及4-DAMP组(2 mg·kg-1·d-1),建立荷瘤模型。连续给药21 d后处死小鼠,计算抑制率、脾指数。实时定量PCR (quantitative reverse-transcription polymerase chain reaction, qRT-PCR)检测A375细胞系中M3R、MIA(melanoma inhibitory activity)、SP-1 (transcription factor Sp1)、Lnc-SPRY4-IT1(SPRY4 Intronic Transcript 1)的基因表达。Western Blot检测ERK1/2(extracellular signal-regulated kinase 1/2)蛋白含量的表达。结果:MTT及EdU均表明,4-DAMP可明显抑制A375细胞的活力(P<0.01)。动物实验结果表明,5-Fu组与4-DAMP组肿瘤体积及质量均明显减小(P<0.05);H&E染色可见细胞间隙变大,结缔组织纤维增多。qRT-PCR结果表明,4-DAMP或转染si-M3R均可降低细胞内MIA、SP-1以及LncRNA-SPRY4-IT1的表达。Western Blot结果表明,4-DAMP或转染si-M3R后均可降低细胞内ERK1/2的表达。结论:M3受体拮抗剂4-DAMP在细胞及动物实验中均能抑制黑色素瘤增殖发挥抗肿瘤功效,降低肿瘤细胞内MIA、SP-1的mRNA和LncRNA-SPRY4-IT1的表达,同时抑制了ERK1/2的表达。

Objective: The effect of M3 receptor antagonist 4-DAMP(4-diphenylacetoxy-N-methylpiperidine-methiodide) on the proliferation of melanoma wasexplored, 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 establish tumor-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. 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 M3 R、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-M3 R transfection. Compared with the control group, the expression of ERK1/2 was decreased by 4-DAMP or si-M3 R 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.