人乳头瘤病毒(HPV)是几乎所有宫颈癌形式的病原体，它可以在病毒融入宿主基因组和同时失去病毒调控基因E2的情况下产生。基因为基础的传递方法已经表明，E2重引入可在体外减少增殖能力并促进凋亡。本研究探讨我们的钙依赖性蛋白质基于的传递系统TAT-CaM是否可以将功能性E2蛋白直接传递到宫颈癌细胞中，限制增殖能力并诱导细胞死亡。从大肠杆菌中表达和提纯了含有CaM结合序列(CBS-E2)的HPV16 E2蛋白和TAT-CaM。生物层干涉法验证了钙依赖性结合动力学。等摩尔TAT-CaM:CBS-E2构件被传递到HPV16+ SiHa细胞系中，并通过共聚焦显微镜验证了摄取。增殖能力通过MTS测定，细胞死亡通过乳酸脱氢酶释放测定。人微血管细胞(HMECs)被用作对照。预期地，在钙存在时，TAT-CaM与CBS-E2高亲和力结合，并在去除钙离子时迅速解离。通过TAT-CaM引入，荧光标记的CBS-E2在细胞内部通过在核深度处进行正交投影被检测出来。在分裂的细胞中，E2被重新定位到与有丝分裂纺锤体相关的区域。接受每日CBS-E2剂量4天的细胞在低剂量下显示出显著的代谢活性降低和高剂量下的细胞死亡，与对照组相比。这种表型在7天后无需进一步处理就被保留下来。在第12天进行亚培养时，治疗细胞恢复了它们的增殖能力。利用TAT-CaM平台，生物活性的E2蛋白被传递到活着的宫颈癌细胞中，以时间和剂量依赖的方式诱导衰老和细胞死亡。这些结果表明，这种无核酸和无病毒的传递方法可被利用开发新型的有效蛋白质治疗剂。© 2023作者。由Wiley Periodicals公司出版的《癌症报告》。

Human papillomavirus (HPV) is the causative agent of nearly all forms of cervical cancer, which can arise upon viral integration into the host genome and concurrent loss of viral regulatory gene E2. Gene-based delivery approaches show that E2 reintroduction reduces proliferative capacity and promotes apoptosis in vitro.This work explored if our calcium-dependent protein-based delivery system, TAT-CaM, could deliver functional E2 protein directly into cervical cancer cells to limit proliferative capacity and induce cell death.TAT-CaM and the HPV16 E2 protein containing a CaM-binding sequence (CBS-E2) were expressed and purified from Escherichia coli. Calcium-dependent binding kinetics were verified by biolayer interferometry. Equimolar TAT-CaM:CBS-E2 constructs were delivered into the HPV16+ SiHa cell line and uptake verified by confocal microscopy. Proliferative capacity was measured by MTS assay and cell death was measured by release of lactate dehydrogenase. As a control, human microvascular cells (HMECs) were used. As expected, TAT-CaM bound CBS-E2 with high affinity in the presence of calcium and rapidly disassociated upon its removal. After introduction by TAT-CaM, fluorescently labeled CBS-E2 was detected in cellular interiors by orthogonal projections taken at the depth of the nucleus. In dividing cells, E2 relocalized to regions associated with the mitotic spindle. Cells receiving a daily dose of CBS-E2 for 4 days showed a significant reduction in metabolic activity at low doses and increased cell death at high doses compared to controls. This phenotype was retained for 7 days with no further treatments. When subcultured on day 12, treated cells regained their proliferative capacity.Using the TAT-CaM platform, bioactive E2 protein was delivered into living cervical cancer cells, inducing senescence and cell death in a time- and dose-dependent manner. These results suggest that this nucleic acid and virus-free delivery method could be harnessed to develop novel, effective protein therapeutics.© 2023 The Authors. Cancer Reports published by Wiley Periodicals LLC.