BACKGROUND New therapies are urgently needed in melanoma particularly in late-stage patients not responsive to immunotherapies and kinase inhibitors. METHODS Drug screening, IC50 determinations as well as synergy assays were detected by the MTT assay. Apoptosis using Annexin V and 7AAD staining was assessed using flow cytometry. TUNEL staining was performed using immunocytochemistry. Changes in phosphorylation of key molecules in PI3K/Akt/mTOR and other relevant pathways were detected by western blot as well as immunocytochemistry. To assess in vivo anti-tumor activity of Tegaserod, syngeneic intravenous and subcutaneous melanoma xenografts were used. Immunocytochemical staining was performed to detect expression of active Caspase-3, cleaved Caspase 8 and p-S6 in tumors. Evaluation of immune infiltrates was carried out by flow cytometry. RESULTS Using a screen of 770 pharmacologically active and/or FDA approved drugs, we identified Tegaserod (Zelnorm, Zelmac) as a compound with novel anti-cancer activity which induced apoptosis in murine and human malignant melanoma cell lines. Tegaserod (TM) is a serotonin receptor 4 agonist (HTR4) used in the treatment of irritable bowel syndrome (IBS). TM's anti-melanoma apoptosis-inducing effects were uncoupled from serotonin signaling and attributed to PI3K/Akt/mTOR signaling inhibition. Specifically, TM blunted S6 phosphorylation in both BRAFV600E and BRAF wildtype (WT) melanoma cell lines. TM decreased tumor growth and metastases as well as increased survival in an in vivo syngeneic immune-competent model. In vivo, TM also caused tumor cell apoptosis, blunted PI3K/Akt/mTOR signaling and decreased S6 phosphorylation. Furthermore TM decreased the infiltration of immune suppressive regulatory CD4+CD25+ T cells and FOXP3 and ROR-γt positive CD4+ T cells. Importantly, TM synergized with Vemurafenib, the standard of care drug used in patients with late stage disease harboring the BRAFV600E mutation and could be additively or synergistically combined with Cobimetinib in both BRAFV600E and BRAF WT melanoma cell lines in inducing anti-cancer effects. CONCLUSION Taken together, we have identified a drug with anti-melanoma activity in vitro and in vivo that has the potential to be combined with the standard of care agent Vemurafenib and Cobimetinib in both BRAFV600E and BRAF WT melanoma.

中文翻译：

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重新使用5-羟色胺激动剂Tegaserod作为黑色素瘤的抗癌药：分子机制和临床意义。

背景技术在黑素瘤中，特别是在对免疫疗法和激酶抑制剂无反应的晚期患者中，迫切需要新的疗法。方法采用MTT法检测药物筛选，IC50测定及协同作用。使用流式细胞术评估使用Annexin V和7AAD染色的细胞凋亡。使用免疫细胞化学进行TUNEL染色。通过蛋白质印迹和免疫细胞化学检测PI3K / Akt / mTOR和其他相关途径中关键分子的磷酸化变化。为了评估替加色罗的体内抗肿瘤活性，使用了同系静脉内和皮下黑色素瘤异种移植物。进行免疫细胞化学染色以检测肿瘤中活性Caspase-3，裂解的Caspase 8和p-S6的表达​​。通过流式细胞术进行免疫浸润的评估。结果使用770种具有药理活性和/或FDA批准的药物的筛选，我们确定Tegaserod（Zelnorm，Zelmac）为具有新型抗癌活性的化合物，可诱导鼠和人恶性黑色素瘤细胞系的凋亡。Tegaserod（TM）是用于治疗肠易激综合症（IBS）的血清素4受体激动剂（HTR4）。TM的抗黑素瘤凋亡诱导作用与5-羟色胺信号传导无关，并归因于PI3K / Akt / mTOR信号传导抑制。具体而言，TM使BRAFV600E和BRAF野生型（WT）黑色素瘤细胞系中的S6磷酸化减弱。在体内同基因免疫能力模型中，TM减少了肿瘤的生长和转移，并增加了存活率。在体内，TM还引起肿瘤细胞凋亡，使PI3K / Akt / mTOR信号减弱，S6磷酸化降低。此外，TM减少了免疫抑制性调节性CD4 + CD25 + T细胞以及FOXP3和ROR-γt阳性CD4 + T细胞的浸润。重要的是，TM与Vemurafenib协同作用，后者是具有BRAFV600E突变的晚期疾病患者使用的护理药物标准，可以在BRAFV600E和BRAF WT黑色素瘤细胞系中与Cobimetinib加成或协同结合，以诱导抗癌作用。结论综上所述，我们已经确定了一种在体外和体内均具有抗黑素瘤活性的药物，该药物有可能在BRAFV600E和BRAF WT黑素瘤中与维拉非尼和Cobimetinib的标准治疗药物联合使用。带有BRAFV600E突变的晚期疾病患者使用的护理药物标准，可以在BRAFV600E和BRAF WT黑色素瘤细胞系中与Cobimetinib加成或协同结合，以诱导抗癌作用。结论综上所述，我们已经确定了一种在体外和体内均具有抗黑素瘤活性的药物，该药物有可能在BRAFV600E和BRAF WT黑素瘤中与维拉非尼和Cobimetinib的标准治疗药物联合使用。具有BRAFV600E突变的晚期疾病患者使用的护理药物标准，可以在BRAFV600E和BRAF WT黑色素瘤细胞系中与Cobimetinib加成或协同结合，以诱导抗癌作用。结论综上所述，我们已经确定了一种在体外和体内均具有抗黑素瘤活性的药物，该药物有可能在BRAFV600E和BRAF WT黑素瘤中与维拉非尼和Cobimetinib的标准治疗药物联合使用。