The Effects Of NTSR1Antagonist SR48692on The Roliferation, Apoptosis Of Melanoma Cells

Objective: Malignant melanoma is a highly malignant melanoma with apropensity for distant metastasis resulting in poor prognosis, which originatedin the neural crest cell. In recent years, the incidence of malignant melanomaincreases rapidly in China. The majority of patients with early-stagemelanoma can be cured by surgery, but for the advanced tumor, the standardchemotherapeutic approaches used in the treatment have been disappointing.So to explore new chemotherapeutic regimens with high efficacy and lowtoxicity is particularly needed.The neurotensin is a single-chain polypeptide consisting of13aminoacids, which can regulate gastrointestinal digestive function through theneuroendocrine and the role of hormones. We have found that neurotensin canpromote the proliferation of some tumor cells. Neurotensin receptor subtype1(NTSR1), coupling with G protein, is a high-affinity receptor for neurotensin.A variety of biological effects of NT are mediated by the specific binding ofNT and NTSR1，indentification its crucial role in tumor progression. It hasbeen found that the expression of NT and NTSR1in some tumor cells wassignificantly higher than in normal tissue. SR48692, NT receptor competitiveantagonist, can block the combination of NT and NTSR1specifically. It hasbeen demonstrated that SR48692could inhibit the role of NT in promotingtumor growth. Therefore we consider that NT/NTSR1is promising to conductas a new tumor diagnostic indicator and therapeutic target, at the same time,SR48692is a potential drug for tumor therapy. In order to find new diagnosticindicators and effective therapeutic drugs for melanoma, this studyinvestigated whether NTSR1was expressed in A375melanoma cells or not,observed the effect of SR48692on proliferation, cell cycle and apoptosis inhuman A375melanoma cells, and tested the condition of SR48692in inhibiting colony formation in soft agar and the tumor growth of tumor-bearingmice in vivo.Methods:1In this study, we detected the expression of NTSR1in A375melanomacells by RT-PCR、Western blot and NTSR1immunofluorescence.2The change on the proliferation capacity of melanoma cells after treatedwith SR48692（5μmol/L、10μmol/L）for24h、48h、72h、96h was determinedby the method of cell counting、MTT. BrdU immunofluorescence was used toreveal the effect of SR48692on the growth of A375cells.3The effect of SR48692on cell cycle was detected by PI staining andflow cytometry. The effect of SR48692on apoptosis was verified by AnnexinV-FITC/PI staining and Hoechst33258staining. In order to explore themechanism of the apoptosis and cycle arrest induced by SR48692.4The effect of tumorigenic capacity of SR48692on A375melanoma cellswas verified by soft agar assay and the tumor formation in vivo of mice.Results:1We found the overexpression of NTSR1in A375melanoma celllines,which was confirmed simultaneously by RT-PCR、Western blot andNTSR1immunofluorescence.2Both the cell counting and MTT assay demonstrated SR48692couldinhibit the proliferation of A375cells at a concentration (5μmol/L,10μmol/L)and time-dependent effect significantly. BrdU immunofluorescence stainingfurther confirmed that compared to the control group (26.07±2.59%), thepercentage of BrdU-positive cells (7.55±2.76%, P=0.000) of the group treatedwith SR48692reduced significantly.3The flow cytometry show a significant increase in the proportion ofcells in the S phase and G2/M phases, and a significant decrease in the G0/G1phases.We found the representative cell apoptosis morphological changes wasfound in SR48692group through Hoechst33258staining. PI staining flowcytometry show significant apoptosis peak. Annexin V FITC/propidium iodidestaining and flow cytometry show the percentage of apoptosis ingreased (63.24±1.26%, P=0.000), compared with vehicle-treated cells (7.35±0.64%).4Soft agar colony assay showed that compared with the control group(55.67±9.01), SR48692inhibited clone formation significantly (24.33±5.86, P=0.005). The study of A375melanoma cells in vivo xenografted in theNOD/SCID mice model showed that the volume and weight of xenografttumor were remarkable decreased after treated with SR48692(0.117+0.023g),compared with control group(0.243+0.022g,P<0.05).Conclusion: We detected the expression of NTSR1in melanoma cellsand found the remarkable expression of NTSR1in melanoma cell line.Theresult revealed that NT/NTSR1pathway was activated in A375melanoma cellline.In this study, we found that SR48692could inhibit A375melanoma cellsproliferation significantly by inducing S phase, G2/M phase arrest andinducing apoptosis, as well as inhibit colony formation and tumorigeniccapacity in vivo. Considering the striking inhibition effect on melanoma cells,SR48692represents a potential new candidate for targeted therapy inmelanoma.