Potential of new anti-cancer agents targeting the nuclear translocation signaling of HB-EGF C-terminal fragments

目前对晚期结直肠癌的治疗靶点主要集中在表皮生长因子受体(EGFR)信号转导，但其与化疗的相加作用仍然有限。去整合素和金属蛋白酶(ADAM)可裂解前肝素结合的表皮生长因子样生长因子(proHB-EGF)。可溶性HB-EGF可激活EGFR。同时，ProHB-EGF的羧基末端片段(HB-EGF-CTF)移位到核内核膜，通过结合转录抑制因子锌指蛋白(PLZF)调控细胞增殖，从而导致其核输出。我们推测，抑制Hb-EGF-CTF核转位可能是阻止细胞proliferation.Methods:12-O-tetradecanoylphorbor-13-acetate(TPA)激活ADAM的一种新策略。筛选出9000种化合物阻断Hb-EGF-CTF与早幼粒细胞…的结合Alphascreen系统更多的白血病锌指(PLZF)。然后将获得的候选基因用于阻断HB-EGF-CTF与结肠癌细胞HT29和HCT116中PLZF的结合。生长曲线分析法检测细胞增殖情况。免疫荧光染色、免疫沉淀和Western blotting分别检测HB-EGF-CTF和PLZF在细胞内的定位和相互作用。此外，还研究了在血管紧张素II 1型受体(AT1R)基因敲除过程中，所获得的候选基因对EGFR磷酸化、HB-EGF-CTF核转位和PLZF输出的影响。结果：替米沙坦和坎地沙坦是潜在的候选药物。替米沙坦对TPA诱导的细胞增殖的抑制作用强于坎地沙坦。替米沙坦但不能阻断TPA刺激时HB-EGF-CTF的核转位和HB-EGF-CTF与PLZF的结合。替米沙坦和坎地沙坦均不能抑制TPA诱导的EGFR磷酸化，而替米沙坦可抑制TPA诱导的AT1R下调后HB-EGF-CTF的核转位。结论：替米沙坦抑制HB-EGF-CTF核转位可能是一种防止细胞增殖的新策略。较少

Current treatment target toward advanced colorectal cancers is mainly focused on the epidermal growth factor receptor (EGFR) signaling, but its additive effects with chemotherapy are still limited. A disintegrin and metalloproteinase (ADAM) cleaves the proheparin-binding epidermal growth factor like growth factor (proHB-EGF). And soluble HB-EGF activates EGFR. In parallel, the carboxy-terminal fragment of proHB-EGF (HB-EGF-CTF) translocate into the inner nuclear membrane, subsequently exerts on the regulation of cell proliferation by binding nuclear promyelocytic leukemia zinc finger (PLZF) protein, a transcriptional repressor, thereby causing its nuclear export. We hypothesized that the inhibition of HB-EGF-CTF nuclear translocation may be a new strategy in preventing cell proliferation.Methods:12-O-tetradecanoylphorbor-13-acetate (TPA) was treated to activate ADAM. Nine-thousand chemical compounds were screened for their efficacies in blocking the binding of HB-EGF-CTF to promyelocyt … More ic leukemia zinc finger (PLZF) with Alphascreen system. The obtained candidates were then used to block the binding of HB-EGF-CTF to PLZF in colon cancer cells, HT29 and HCT116. Cell proliferation was investigated with a growth curve assay. The intracellular localization, and association between HB-EGF-CTF and PLZF, was assessed with immunofluorescent staining, and immunoprecipitation and Western blotting, respectively. The effects of obtained candidates on EGFR phosphorylation and on nuclear translocation of HB-EGF-CTF and export of PLZF during the angiotensin II type1 receptor (AT1R) knockdown were also investigated. Results: Telmisartan and candesartan were found to be potential candidates. Telmisartan inhibited TPA-induced cell proliferation stronger than candesartan. Telmisartan, but not candesartan blocked the nuclear translocation of HB-EGF-CTF, and binding of HB-EGF-CTF to PLZF, during TPA stimulation. Both telmisartan and candesartan did not inhibit TPA-induced EGFR phosphorylation, and telmisartan, but not candesartan, inhibited TPA-induced nuclear translocation of HB-EGF-CTF after knockdown of AT1R. Conclusions: The inhibition of HB-EGF-CTF nuclear translocation with telmisartan may be a novel strategy in preventing cell proliferation. Less