Utilization of a cell-penetrating peptide-adaptor for delivery of human papillomavirus protein E2 into cervical cancer cells to arrest cell growth and promote cell death

• Type of Material: Article
• Authors: Julia LeCher, Emory University; Hope L Didier, Kennesaw State University; Robert L Dickson, Kennesaw State University; Lauren R Slaughter, Kennesaw State University; Juana C Bejarano, Kennesaw State University; Steven Ho, Kennesaw State University; Scott J Nowak, Kennesaw State University; Carol A Chrestensen, Kennesaw State University; Jonathan L McMurry, Kennesaw State University
• Language: English
• Date: 2023-05-01
• Publisher: Emory University Libraries
• Publication Version: Final Published Version
• Copyright Statement: © 2023 The Authors. Cancer Reports published by Wiley Periodicals LLC.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1002/cnr2.1810
• Title of Journal or Parent Work: Cancer Reports
• Volume: 6
• Issue: 5
• Start Page: e1810
• End Page: e1810
• Grant/Funding Information: This work was also supported by Public Health Service grants R16 GM 145448 and R15 HL 161738 to Scott J. Nowak. Hope L. Didier was supported by a Birla Carbon Fellowship from Kennesaw State University College of Science & Mathematics, Kennesaw, GA. Juana C. Bejarano was supported by a Mentor‐Protegee grant from Kennesaw State University College of Science & Mathematics, Kennesaw, GA.; This work was primarily funded by Public Health Service grant R15 EB028609.
• Keywords: Papillomavirus E7 Proteins; E6; HPV-16; cell-penetrating peptides; Calcium; Female; E2; Apoptosis; Cell-Penetrating Peptides; Humans; Uterine Cervical Neoplasms; E7; Human Papillomavirus Viruses; cervical cancer
• Research Categories: Chemistry, Biochemistry; Biology, Cell

Abstract

Background: 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. Aims: 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. Materials and Results: 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. Conclusions: 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.