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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
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  • Julia LeCher,
  • Hope Didier,
  • Robert Dickson,
  • Lauren Slaughter,
  • J. Bejarano,
  • Steven Ho,
  • Scott Nowak,
  • Carol Chrestensen,
  • Jonathan McMurry
Julia LeCher
Emory University School of Medicine

Corresponding Author:[email protected]

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Hope Didier
Kennesaw State University
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Robert Dickson
Kennesaw State University
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Lauren Slaughter
Kennesaw State University
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J. Bejarano
Kennesaw State University
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Steven Ho
Kennesaw State University
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Scott Nowak
Kennesaw State University
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Carol Chrestensen
Kennesaw State University
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Jonathan McMurry
Kennesaw State University
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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. 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. Methods: TAT-CaM and the HPV16 E2 protein containing a CaM-binding sequence (CBS-E2) were expressed and purified from E. 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 for specificity to HPV+ cells, human microvascular cells (HMECs) were used. Results: TAT-CaM bound CBS-E2 with high affinity in the presence of calcium and rapidly disassociated in its absence. After introduction by TAT-CaM, E2 was detected in cellular interiors by orthogonal projects taken at the depth of the nucleus. In dividing cells, E2 relocalized to regions associated with the mitotic spindle. Cells receiving a single daily dose of CBS-E2 for 4 days showed a significant reduction in metabolic activity at low doses and cell death at high doses compared to controls. This phenotype was retained for 7 days with no further treatments. When subcultured at 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.
16 Nov 2022Submitted to Cancer Reports
18 Nov 2022Submission Checks Completed
18 Nov 2022Assigned to Editor
18 Nov 2022Review(s) Completed, Editorial Evaluation Pending
12 Dec 2022Reviewer(s) Assigned
24 Jan 2023Editorial Decision: Revise Major
21 Feb 20231st Revision Received
22 Feb 2023Assigned to Editor
22 Feb 2023Submission Checks Completed
22 Feb 2023Review(s) Completed, Editorial Evaluation Pending
23 Feb 2023Reviewer(s) Assigned
07 Mar 2023Editorial Decision: Revise Minor
08 Mar 20232nd Revision Received
09 Mar 2023Assigned to Editor
09 Mar 2023Submission Checks Completed
09 Mar 2023Review(s) Completed, Editorial Evaluation Pending
12 Mar 2023Editorial Decision: Accept