BRN2转录因子与恶性黑色素瘤的发生发展有关。使用来自BRN2转录因子POU结构域的合成肽R18H确定了针对B16F10-Nex2细胞的细胞毒性活性和细胞死亡机制。
确定来自BRN2转录因子的POU结构域的肽R18H对B16F10-Nex2细胞的细胞死亡机制和体内活性。
通过MTT方法测定细胞活力。用B16F10-Nex2细胞攻击C57Bl/6小鼠并用R18H处理。为了确定细胞死亡的类型,我们用了TUNEL法,膜联蛋白V和PI,Hoechst,DHE,并测定caspase的激活和细胞色素c的释放。进行透射电子显微镜检查以验证肽处理后的形态改变。
肽R18H在治疗的第一个小时显示出抗肿瘤活性,并计算2和24小时的EC50%,为0.76±0.045mM和0.559±0.053mM,分别。24h后细胞凋亡明显,基于DNA降解,染色质凝聚,增加超氧阴离子的产生,磷脂酰丝氨酸易位,在B16F10-Nex2细胞中激活半胱天冬酶3和8,并释放细胞外细胞色素c。肽的细胞毒活性不受坏死抑制剂的影响,并且处理的细胞在细胞外培养基中不释放LDH。此外,在用肽R18H处理后观察到体内抗肿瘤活性。
来自BRN2转录因子的肽R18H在B16F10-Nex2中诱导凋亡并在体内显示抗肿瘤活性。
BRN2 transcription factor is associated with the development of malignant melanoma. The cytotoxic activities and cell death mechanism against B16F10-Nex2 cells were determined with synthetic peptide R18H derived from the POU domain of the BRN2 transcription factor.
To determine the cell death mechanisms and in vivo activity of peptide R18H derived from the POU domain of the BRN2 transcription factor against B16F10-Nex2 cells.
Cell viability was determined by the MTT method. C57Bl/6 mice were challenged with B16F10-Nex2 cells and treated with R18H. To identify the type of cell death, we used TUNEL assay, Annexin V and PI, Hoechst, DHE, and determination of caspase activation and cytochrome c release. Transmission electron microscopy was performed to verify morphological alterations after peptide treatment.
Peptide R18H displayed antitumor activity in the first hours of treatment and the EC50% was calculated for 2 and 24h, being 0.76 ± 0.045 mM and 0.559 ± 0.053 mM, respectively. After 24h apoptosis was evident, based on DNA degradation, chromatin condensation, increase of superoxide anion production, phosphatidylserine translocation, activation of caspases 3 and 8, and release of extracellular cytochrome c in B16F10-Nex2 cells. The peptide cytotoxic activity was not affected by necroptosis inhibitors and treated cells did not release LDH in the extracellular medium. Moreover, in vivo antitumor activity was observed following treatment with peptide R18H.
Peptide R18H from BRN2 transcription factor induced apoptosis in B16F10-Nex2 and displayed antitumor activity in vivo.