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Apoptosis induced by an antagonist peptide against HPV16 E7 in vitro and in vivo via restoration of p53

机译:HPV16 E7拮抗肽通过恢复p53在体内和体外诱导的细胞凋亡

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Human papilloma virus type 16 (HPV16) E7 is a viral oncoprotein that is believed to play a major role in cervical neoplasia. A novel antagonist peptide against HPV16 E7 was previously selected by phage display screening and the selected peptide was found to have anti-tumor efficacy against HPV16-positive cervical carcinoma through induction of cell cycle arrest. In the current study, to further elucidate the mechanisms of the antagonist peptide, the effects of the peptide on apoptosis are investigated by RT-PCR, Western blotting, MTT assay, TUNEL staining, Annexin V apoptosis assay, flow cytometry, and animal experiments. The antagonist peptide showed obvious anti-tumor efficacy through apoptosis induction, both in HPV16-positive cervical cancer cell lines and tumor xenografts. Our results also revealed that the peptide induced accumulation of cellular p53 and p21, and led to HPV16 E7 protein degradation. In the case of mRNA levels, it resulted in unaltered p53 and HPV16 E7 expression, but increased expression of p21. In contrast, the induction of apoptosis and p53 reactivation effects by the selected peptide were abolished after E7 knocked down with siRNA. These results demonstrate that the selected peptide can induce E7 degradation and lead to marked apoptosis in HPV16-related cancer cells by activating cellular p53 and its target genes, such as p21. Furthermore, the evident therapeutic efficacy obtained from the subcutaneous tumor model experiments in nude mice suggests a therapeutic potential for HPV16-related cancers of the selected peptide. Therefore, this specific peptide may be used to create specific biotherapies for the treatment of HPV 16-positive cervical cancers.
机译:16型人乳头瘤病毒(HPV16)E7是一种病毒癌蛋白,据信在宫颈肿瘤中起主要作用。先前通过噬菌体展示筛选选择了针对HPV16 E7的新型拮抗肽,并发现所选肽通过诱导细胞周期停滞而具有抗HPV16阳性宫颈癌的抗肿瘤功效。在当前的研究中,为进一步阐明拮抗剂肽的机制,通过RT-PCR,Western印迹,MTT测定,TUNEL染色,膜联蛋白V凋亡测定,流式细胞术和动物实验研究了肽对凋亡的影响。在HPV16阳性宫颈癌细胞系和肿瘤异种移植物中,拮抗肽均通过凋亡诱导显示出明显的抗肿瘤功效。我们的结果还表明,该肽诱导细胞p53和p21的积累,并导致HPV16 E7蛋白降解。就mRNA水平而言,它导致p53和HPV16 E7表达不变,但p21表达增加。相反,用siRNA敲除E7后,所选肽对细胞凋亡的诱导作用和p53的再激活作用被取消。这些结果表明,选定的肽可以通过激活细胞p53及其靶基因(例如p21)诱导HPV16相关癌细胞中E7降解并导致明显的细胞凋亡。此外,从裸鼠的皮下肿瘤模型实验中获得的明显治疗功效表明了所选肽与HPV16相关的癌症的治疗潜力。因此,该特异性肽可用于创建用于治疗HPV 16阳性宫颈癌的特异性生物疗法。

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