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
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.