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Citation: Ali, U.; Bette, M.; Ambreen,
G.; Pinnapireddy, S.R.; Tariq, I.;
Marquardt, A.; Stuck, B.A.;
Bakowsky, U.; Mandic, R.
RNAi-Mediated Knockdown of
Cottontail Rabbit Papillomavirus
Oncogenes Using Low-Toxicity
Lipopolyplexes as a Paradigm to
Treat Papillomavirus-Associated
Cancers. Pharmaceutics 2023,15, 2379.
https://doi.org/10.3390/
pharmaceutics15102379
Academic Editors: Kenneth K. W. To
and Jun Dai
Received: 25 July 2023
Revised: 25 August 2023
Accepted: 19 September 2023
Published: 25 September 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
pharmaceutics
Article
RNAi-Mediated Knockdown of Cottontail Rabbit
Papillomavirus Oncogenes Using Low-Toxicity Lipopolyplexes
as a Paradigm to Treat Papillomavirus-Associated Cancers
Uzma Ali 1, 2, †, Michael Bette 3 ,† , Ghazala Ambreen 1,2,† , Shashank R. Pinnapireddy 1,4 , Imran Tariq 1,5 ,
AndréMarquardt 6, Boris A. Stuck 2, Udo Bakowsky 1, *,‡ and Robert Mandic 2 ,*, ‡
1Department of Pharmaceutics and Biopharmaceutics, Philipps-Universität Marburg,
35037 Marburg, Germany; imran.pharmacy@pu.edu.pk (I.T.)
2Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Marburg,
Philipps-Universität Marburg, 35043 Marburg, Germany
3Institute of Anatomy and Cell Biology, Philipps-Universität Marburg, 35037 Marburg, Germany
4CSL Behring Innovation GmbH, 35041 Marburg, Germany
5Punjab University College of Pharmacy, University of the Punjab, Lahore 54590, Pakistan
6Department of Pathology, Klinikum Stuttgart, 70174 Stuttgart, Germany
*Correspondence: udo.bakowsky@staff.uni-marburg.de (U.B.); mandic@med.uni-marburg.de (R.M.);
Tel.: +49-6421-2825884 (U.B.); +49-6421-5861400 (R.M.)
†These authors contributed equally to this work.
‡These authors contributed equally to this work.
Abstract:
The cottontail rabbit papillomavirus (CRPV)-associated VX2 carcinoma of the New Zealand
White rabbit serves as a model system for human papillomavirus (HPV)-associated head and neck
squamous cell carcinomas (HNSCCs). The aim of this study was to evaluate the tumor-inhibiting
effect of RNAi-mediated knockdown of the CRPV oncogenes, E6 and E7, using siRNA-loaded
lipopolyplexes (LPPs). VX2-carcinoma-derived cells were cultured for up to 150 passages. In
addition, CRPV E6 and E7 oncogenes were transiently expressed in COS-7 cells. Efficiency and
safety of LPPs were evaluated in both VX2 cells and the COS-7 cell line. Both of these
in vitro
CRPV
systems were validated and characterized by fluorescence microscopy, Western blot, and RT-qPCR.
Efficient knockdown of CRPV E6 and E7 was achieved in VX2 cells and COS-7 cells pretransfected
with CRPV E6 and E7 expression vectors. Knockdown of CRPV oncogenes in VX2 cells resulted in
reduced viability, migration, and proliferation and led to a G0/G1 block in the cell cycle. CRPV E6
and E7 siRNA-loaded LPPs could represent promising therapeutic agents serving as a paradigm
for the treatment of papillomavirus-positive cancers and could be of value for the treatment of
CRPV-associated diseases in the rabbit such as papillomas and cancers of the skin.
Keywords:
head and neck cancer; HNSCC; VX2 carcinoma; papillomavirus; CRPV; E6; E7;
lipopolyplexes; RNAi
1. Introduction
Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common
cancer, being responsible for 1–2% of all cancer deaths [
1
,
2
]. Approximately 50% of HNSCC
patients have a 5-year life expectancy of 10–40% [
3
]. High-risk human papillomavirus
(HPV) has been implicated in the pathogenesis of HNSCCs, particularly oropharyngeal
cancer. For more than two decades, the auricular VX2 carcinoma of the New Zealand
White (NZW) rabbit serves as an animal model for HNSCC [
4
,
5
] and is deployed in the
evaluation of novel anticancer therapies as well as diagnostic procedures [6]. Advantages
of this model are the ease of transplantation, rapid tumor growth, and the presence of
local and distant metastatic spread. VX2 tumor development is associated with the cot-
tontail rabbit papillomavirus (CRPV) and was first characterized in 1933 by Shope and
Pharmaceutics 2023,15, 2379. https://doi.org/10.3390/pharmaceutics15102379 https://www.mdpi.com/journal/pharmaceutics
Pharmaceutics 2023,15, 2379 2 of 18
Hurst [
7
]; therefore, it is also known as the Shope papillomavirus. CRPV-induced rabbit
papillomas are comparable in etiology and mechanism to many naturally occurring lesions
induced by HPV [
8
,
9
]. CRPV, like HPV, induces papillomatosis, possibly leading to the
development of squamous cell carcinomas. The metastatic pattern of VX2 tumors mim-
ics the natural pattern seen in HPV-associated human HNSCC [
10
]. In this context, the
VX2 model helps us to better understand HPV-associated tumors. VX2 carcinoma cells,
therefore, can be considered as an equivalent to HPV-positive HNSCC cells. From this
perspective, it is of paramount importance to establish a papillomavirus-positive VX2 cell
line which would allow us to perform
in vitro
studies, thereby helping to reduce animal
experiments according to the 3R principle [
11
]. Several groups reported the generation of
VX2-carcinoma-derived cell lines such as VX7, VX-T, and VX-R. However, these cell lines
exhibited fewer anaplastic characteristics, loss of transplantability in the host, or loss of abil-
ity to establish VX2 tumors [
12
–
16
]. At the moment, VX2 carcinoma is available as frozen
tissue or a serially transplantable VX2 tumor suspension [
17
]. Notably, HPV-positive cell
lines are underrepresented in numbers, likely due to their lower survival rate in culture as
compared to HPV-negative cell lines, which limits studies aiming to understand the biology
of papillomavirus-associated tumors [
18
]. Since the VX2 carcinoma rabbit tumor serves
as a model system for HNSCC, we were interested in establishing a VX2-tumor-derived
cell line as well as a CRPV E6/E7 transiently expressing cell model to provide a platform
for the design and development of antiviral therapies and to comply with the 3R rule. For
the latter one, we deployed the widely used monkey-kidney-derived cell line COS-7 [
19
].
CRPV encodes two types of E6 proteins, a short (SE6) and a long (LE6) version which is
equivalent to the E6 protein expressed by HPV-16 and HPV-18. It was observed that CRPV
E6 (SE6 and LE6) does not interact with the E3 ligase E6AP and p53 and therefore cannot
initiate p53 degradation. Recently, it has been shown that CRPV and HPV-38 E6 interact
with p300 (a histone acetyltransferase), which inhibits p53-mediated apoptosis [
20
]. Studies
reported that the CRPV E7 protein interferes with different functions of the retinoblastoma
protein, as seen for HPV-16 and HPV-18 [
21
]. Similarly to that observed for HPV-associated
HNSCC tumors, CRPV-related oncogenes E6 and E7 are implicated in viral replication,
transformation, tumor growth, and progression, and thus represent potential targets for
therapeutic approaches [
22
]. Thus, in this study, particular emphasis is laid on CRPV E6
and E7 as therapeutic targets. Therefore, siRNA-mediated knockdown of these oncogenes
is evaluated as a treatment option for CRPV-positive carcinomas. To achieve successful
and efficient gene knockdown, the most crucial step is the selection of appropriate, most
effective, and safe gene delivery systems. In recent years, much attention has been given to
nonviral delivery systems since they elicit fewer immune responses while enabling unre-
stricted packaging capacity and ease of synthesis as well as cost-effectiveness and improved
targeting potential [
23
]. Lipopolyplexes, a second-generation nonviral gene delivery system
with a size range of 100–200 nm, possess high transfection efficiencies, stability, and im-
proved biocompatibility [
24
]. Lipopolyplex formulations (DOPE:DPPC:cholesterol) possess
higher transfection efficiencies in various cancer cell lines [
25
]. PEI (polyethylenimine)-
based lipopolyplexes [
26
] were used as transfecting reagents for transient transfections.
One main aim of this study was to elucidate the cellular uptake and cytotoxicity level of
lipopolyplexes in VX2 and COS-7 cells. Transiently transfected cells were then used to
study various biological processes associated with short-term gene expression or gene
inhibition (RNAi-mediated gene silencing). This study therefore evaluates siRNA-loaded
lipopolyplexes as a treatment option for papillomavirus-associated carcinomas.
2. Materials and Methods
2.1. Cells and Cell Culture
The African Green Monkey-derived SV40-transformed kidney fibroblast cell line
COS-7 was cultivated in Dulbecco’s Modified Eagle Medium (DMEM, PAA Laboratories,
Pasching, Austria) supplemented with 10% fetal calf serum (FCS, Sigma Aldrich Chemie
GmbH, Taufkirchen, Germany), 2 mmol/L L-Glutamine, 100 U/mL penicillin/streptomycin
Pharmaceutics 2023,15, 2379 3 of 18
(both Capricorn, Ebsdorfergrund, Germany), 50
µ
g/mL gentamicin, and 50
µ
g/mL ampho-
tericin (both Biochrom, Berlin, Germany) at 37
◦
C, 5% CO
2
, in a humidified atmosphere.
VX2 cells were derived from a solid VX2 tumor that was excised from a tumor-bearing
rabbit as described previously [
18
] and grown in DMEM/Ham’s F-12 medium (Capricorn)
containing 10% FCS (Sigma Aldrich Chemie GmbH) 100 U/mL penicillin/streptomycin
(Capricorn), 50
µ
g/mL gentamicin, and 50
µ
g/mL amphotericin (both Biochrom) in a
humidified atmosphere at 37
◦
C, 5% CO
2
. Surviving cells appeared to be mostly adherent
and were considered to represent mainly VX2 tumor cells since normal cells should have
become senescent. Both cell lines were grown in 100 mm tissue culture dishes and passaged
after reaching 80–90% confluency. To differentiate epithelial VX2-derived tumor cells from
fibroblasts, cells from early (4th to 8th) and late (40th to 50th) passages were investigated
by immunocytochemistry using an antibody directed against the mesenchymal cell marker
vimentin. The nucleus was counterstained with DAPI (1
µ
g/mL in PBS; Roche Diagnostics,
Indianapolis, IN, USA) for 10 min. The signals were documented by confocal laser scanning
microscopy (Leica TCS SP2, Leica Microsystems AG, Wetzlar, Germany).
2.2. Preparation of Lipopolyplexes
Liposomes, polyplexes, and lipopolyplexes (LPPs) were prepared by a previously es-
tablished method [
17
]. Briefly, lipids including DOPE (1,2-Dioleoyl-sn-glycero-3-
phosphoethanolamine), DPPC (1,2-Dipalmitoyl-sn-glycero-3-phosphocholine) (both Lipoid
GmbH, Ludwigshafen, Germany), and cholesterol (Sigma Aldrich Chemie GmbH,
Taufkirchen, Germany), in a proportion of 70:15:15, respectively, were dissolved in
2 mL of a 2:1 (v/v) chloroform: methanol solution in a 5 mL round bottom flask. Once
dissolved, they were evaporated at 40
◦
C on a rotary evaporator (Laborota 4000, Heidolph
Instruments, Schwabach, Germany) equipped with a vacuum pump, to obtain a thin film.
Then, 10 mmol/L HEPES buffer (pH 7.4) was used to hydrate the lipid film by sonication
in a bath sonicator to obtain a homogeneous suspension of liposomes. For further size
reduction, these liposomes were extruded 21 times from an Avanti Mini Extruder by us-
ing polycarbonate membranes (Whatman, Maidstone, UK) of 400 nm and 200 nm pore
size, respectively, followed by filtration through 0.2
µ
m syringe filters. Polyplexes were
prepared with a N/P ratio (ratio of polyethylenimine (PEI) nitrogen atoms to nucleic acid
phosphate atoms) of 9.5. PEI was diluted in 10 mmol/L HEPES buffer and transferred into
1.5 mL reaction tubes containing an equal volume of DNA or siRNA (diluted in 1
×
siRNA
dilution buffer) followed by incubation at RT for 20–25 min. For the preparation of LPPs, a
liposome-to-PEI mass ratio (0.39:1) was used, and calculated amounts of liposomes and
polyplexes were vigorously mixed. Complexes were incubated at RT for 1 h and resulting
LPPs were used for transfection by adding them to the respective cell culture medium.
2.3. RT-qPCR Detection of CRPV E6 and E7 Transcripts
Total RNA from VX2 tumor tissue (positive control) was extracted with the RNeasy
FFPE kit (Qiagen, Hilden, Germany), whereas total RNA from rabbit keratinocytes (negative
control) and VX2 or COS-7 cells was extracted using the RNeasy Mini kit (Qiagen, Germany)
according to the manufacturer’s protocol. RNA concentration was measured with the
NanoDrop ND-1000 system (peqLab Biotechnologie GmbH, Erlangen, Germany). cDNA
was prepared by using 1.0
µ
g of total RNA for each sample deploying the Transcriptor First
Strand cDNA synthesis kit (Roche, Mannheim, Germany) according to the manufacturer’s
protocol. Real-time quantitative PCR analysis (QuantStudio
TM
5 system, Thermo Fisher
Scientific, Waltham, MA, USA) was performed using the PowerUp
TM
SYBR Green Master
Mix (Applied Biosystems, Darmstadt, Germany) according to the manufacturer’s protocol.
The primer length was set between 17 and 27 nt with an optimum length of 20 nt [
19
].
GAPDH,RPL32, and RPLPO of the rabbit were used as housekeeping genes to normalize
gene expression levels of CRPV E6 and CRPV E7 (Supplementary Table S1).
Pharmaceutics 2023,15, 2379 4 of 18
2.4. Western Blot Analysis
SDS PAGE and Western blot analyses were performed under standard conditions.
Whole-cell protein lysates were created through exposing cells to a lysis buffer (20 mmol/L
Tris/HCl pH 7.5; 137 mmol/L NaCl; 10% glycerol; 1% NP40; 2 mmol/L EDTA) supple-
mented with 100
µ
L/mL Protease Inhibitor Cocktail for Mammalian Cell Extracts (cat#
P8340; Sigma-Aldrich Inc., Saint Louis, MO, USA) and 50
µ
L/mL Phosphatase Inhibitor
Cocktail 2 (cat# P5726; Sigma-Aldrich Inc.). Lysis was carried out for 60 min at 4
◦
C, and
the supernatant was harvested after spinning the lysate (>12,000
×
g) for 10 min at 4
◦
C.
Protein concentration was measured with the Bradford method (Bio-Rad Protein Assay Dye
Reagent Concentrate; cat# 5000006; Bio-Rad Laboratories GmbH, Feldkirchen, Germany),
and 20
µ
g of whole-cell protein lysate was separated in a 12% SDS polyacrylamide gel
followed by transfer onto a nitrocellulose membrane. Precision Plus Protein
™
Standards
(Bio-Rad, Hercules, CA, USA) were used as a size control. Subsequently, to detect the
protein of interest, membranes were blocked for 20 min at RT in 3% skim milk/PBS (Merck,
Darmstadt, Germany) followed by overnight incubation with the respective primary anti-
body at 4
◦
C. After overnight incubation, the membranes were washed thrice (10 min each)
in blocking buffer and incubated with an appropriate HRP-coupled secondary antibody
for 1 h at RT. After repeated washing, specific bands were visualized on X-ray film (Agfa,
Cologne, Germany) using the enhanced chemiluminescence (ECL) method (Amersham
Biosciences, Buckinghamshire, UK). Used antibodies were mouse monoclonal anti-
β
-Actin
(clone AC-74, Sigma-Aldrich, Inc., Saint Louis, MO, USA), mouse monoclonal anti-GFP
(clone B-2, sc-9996); rabbit polyclonal anti-RFP (Living colors DsRed Polyclonal antibody,
Takara, Kusatsu, Japan), mouse monoclonal anti-PCNA (clone PC10: sc-56), and mouse
monoclonal anti-vimentin (clone V9, DAKO, Santa Clara, CA, USA). Mouse IgG
κ
light
chain binding protein (m-IgG
κ
BP) conjugated to horseradish peroxidase (HRP) (sc-516102,
1:2000) and goat anti rabbit-IgGk HRP (sc-2004, 1:2000), all from Santa Cruz Biotechnology
(Santa Cruz, CA, USA), were used as secondary antibodies.
2.5. Generation of CRPV E6 and E7 Expression Plasmids
The primers, summarized in Supplementary Table S2, were designed to amplify
the complete coding sequence of CRPV E6 and E7 using mRNA derived from the VX2
tumor [
20
]. For this, total RNA from VX2 tumor tissue was isolated with the RNeasy
Mini kit (Qiagen, Germany) followed by reverse transcription using the Transcriptor First
Strand cDNA synthesis kit (Roche, Mannheim, Germany). PCR was performed using
the REDTaq
®
ReadyMix
TM
PCR Reaction Mix (Sigma-Aldrich, St. Louis, MO, USA) to
generate full-length E6 and E7 cDNA for cloning into the pcDNA3.0, pEGFP-C1, and
pDsRed-monomer-C1 vectors (Invitrogen Corporation, Carlsbad, CA, USA), respectively
(Supplementary Figure S1A). Double restriction enzyme digestion of the purified E6 and
E7 cDNA amplicons and vectors using HindIII/EcoRI (E6, pcDNA3.0), BamHI/EcoRI
(E7, pcDNA3.0), and XhoI/EcoRI (E6 and E7, pEGFP-C1, and pDsRed-monomer-C1) was
carried out followed by gel purification and ligation overnight at 16
◦
C using T4 DNA
ligase (New England Biolabs GmbH, Frankfurt, Germany). Positive plasmids containing
the respective viral cDNA (Supplementary Figure S2B) were further validated by DNA
sequencing (4base lab GmbH, Reutlingen, Germany) and subsequent sequence analysis
(BioEdit software, v 7.0.5.3; Clustal Omega, https://www.ebi.ac.uk/Tools/msa/clustalo/)
(Supplementary Figures S2–S7). Please note that pcDNA3 E6/E7 constructs were generated
but not deployed in the present study. Here, it is noteworthy to point out that CRPV
E7 harbors an internal HindIII site; therefore, BamHI was used for cloning instead. For
plasmid transfection, cells were grown on 6-well plates (1
×
10
6
cells per well) for 24 h
until reaching 60% confluency. Prior to transfection, the cell medium was exchanged with
900
µ
L of serum-free DMEM/Ham’s F-12 medium (Capricorn) for VX2 cells and serum-free
DMEM medium (PAA Laboratories GmbH, Cölbe, Germany) in the case of COS-7 cells,
respectively. Each well received 2
µ
g of plasmid/lipopolyplexes and 800
µ
L of fresh media.
Pharmaceutics 2023,15, 2379 5 of 18
Cells were left incubating for 4 h before adding 1 mL complete media (supplemented with
serum and antibiotics).
2.6. Expression of CRPV E6 and E7 in VX2 and COS-7 Cells
VX2 and COS-7 cells were grown on coverslips in 6-well plates and transfected with
GFP_E6-, GFP_E7-, RFP_E6-, or RFP_E7-expressing plasmids. After incubation for 48 h,
cells were washed thrice with PBS (pH 7.4, containing Ca
2+
and Mg
2+
) and fixed in ice-cold
(
−
20
◦
C) methanol for 5 min. Then, coverslips were incubated for 20 min with 0.1
µ
g/mL
DAPI (Roche Diagnostics, Indianapolis, IN, USA) to counterstain the cell nucleus. Fluores-
cence analysis and documentation was performed by confocal laser scanning microscopy
(Zeiss Axiovert 100M/LSM 510, Carl Zeiss GmbH, Jena, Germany). To evaluate expression
of recombinant proteins, Western blot analysis was performed. Briefly, VX2 and COS-7
cells were seeded on 6-well plates and transfected with green (GFP_E6, GFP_E7) or red
fluorescent protein (RFP_E6, and RFP_E7) tagged E6- or E7-expressing plasmids. The
original pEGFP-C1 and pDsRed-Monomer-C1 vectors were used as a transfection control
(not shown). SDS-PAGE and Western blot analysis were performed as described above.
2.7. MTT Cell Viability Assay
To estimate the extent of cellular toxicity following LPP exposure, VX2 and COS-7 cells
were seeded in 96-well plates at a density of 2
×
10
3
and 4
×
10
3
cells per well, respectively,
and transfected with 0.25
µ
g of plasmid DNA (E7_GFP) per well by either using LPPs or
Lipofectamine
TM
2000 (LF). Forty-eight hours after transfection, plates were washed twice
with PBS (containing Ca
2+
and Mg
2+
) followed by addition of 200
µ
L (2 mg/mL MTT;
Sigma Aldrich Chemie) reagent per well, followed by incubation for additional 4 h to allow
formazan formation. Once formazan crystals were formed, depending on the presence
of viable cells and MTT interaction, medium was removed from the wells and replaced
with 200
µ
L of DMSO. Plates were shaken for 30 min at 120 rpm and the absorbance was
measured at 570 nm in a FLUOStar
TM
Optima plate reader (BMG Labtech, Ortenberg,
Germany) [21].
2.8. RNAi Knockdown of CRPV E6 and E7 by siRNA-Loaded Lipopolyplexes
Three different types of siRNAs were selected from genome sequences of CRPV E6
and E7, as shown in Supplementary Table S3. For siRNA transfection, cells were grown
on 6-well plates (1
×
10
6
cells per well) for 24 h until reaching 60% confluency. Prior to
transfection, the cell medium was changed to 900
µ
L of serum-free DMEM/Ham’s F-12
medium. A total of 100
µ
L of siRNA-loaded LPPs was added to the cells with subsequent
incubation for 4 more hours followed by adding 1 mL complete media (with serum and
antibiotics). After 48 h incubation, cells were trypsinized and pellets were saved at
−
80
◦
C
for RT-qPCR and Western blot analysis. Knockdown efficiency was monitored by RT-qPCR.
The effect of RNAi on cellular viability was evaluated with the MTT assay. To confirm
that siRNAs against CRPV E6 and E7 oncogenes have no major off-target effects and are
suitable for specific gene knockdown, a series of experiments was carried out using the
VX2-unrelated and CRPV-negative cell line COS-7 that is widely deployed in cell biology
studies. COS-7 cells were seeded in 6-well plates at a density of 1
×
10
6
cells per well in
DMEM medium with 10% FCS and antibiotics. After reaching 60–70% confluence, COS-7
cells were transfected with 2
µ
g of CRPV_E6_GFP- or CRPV_E7_GFP-expressing plasmids,
using LPPs as transfecting reagents. After 24 h incubation, fluorescence microscopy (Zeiss
Axiovert 100 M, Carl Zeiss Microscopy GmbH, Jena, Germany) was used to evaluate
transfection efficiency. COS-7 cells were then transfected with CRPV_E6 or CRPV_E7
siRNA using siRNA-loaded LPPs as described above. Nontarget siRNA was used as a
control to account for unspecific “off-target” effects. The expression levels of CRPV_E6
and CRPV_E7 in transiently transfected COS-7 cells were notably higher than in VX2 cells.
Nontarget siRNA was used as a control in the same concentration as that used for E6/E7
specific knockdown. Subsequently, cells were trypsinized and cell pellets were stored for
Pharmaceutics 2023,15, 2379 6 of 18
RT-qPCR and Western blot analysis, respectively. For flow cytometry analysis, COS-7 cells
were fixed in 70% ethanol. RT-qPCR and Western blot analyses were performed using the
same protocols as described above.
2.9. Measuring CRPV E6 and E7 Knockdown in COS-7 Cells by Flow Cytometry
RNAi knockdown of CPRV E6 and E7 in COS-7 cells should also cause a decline in
GFP fluorescence, which serves as a tag for both viral oncoproteins. To evaluate this, GFP
fluorescence in COS-7 cells, transfected with CRPV_E6 and CRPV_E7 siRNA-loaded LPPs,
was measured by flow cytometry. For this, COS-7 cells were fixed in 70% ethanol and
centrifuged for 10 min at 300 xg. After removing the ethanol, the pellet was washed twice
in PBS and resuspended in 500
µ
L PBS followed by flow cytometry analysis (BD LSR II,
Becton Dickinson, Franklin Lakes, New Jersey). Dead cells were excluded from analysis
using forward- and side-scatter parameters. The GFP signal was detected at 530 nm (30 nm
bandwidth filter). Analysis was performed with the FlowJo
™
software (version 7.6.5, Tree
Star Inc., Ashland, OR, USA).
2.10. Wound Healing Assay
VX2 cells were seeded at a density of 1
×
10
6
cells per well in a 6-well plate in
DMEM/Ham’s F-12 medium containing serum and antibiotics. After 24 h, the medium
was replaced with 900
µ
L of serum-free DMEM/Ham’s F-12 medium followed by the
addition of 100
µ
L siRNA-loaded LPPs into each well (nontarget siRNA 50 nmol/L, E6
siRNA 50 nmol/L, E7 siRNA 50 nmol/L, and E6 + E7 siRNA 25 + 25 nmol/L). After 4 h,
1 mL DMEM/Ham’s F-12 medium was added. Twenty-four hours post siRNA transfection,
the layer of cells was scratched by using a 100
µ
L sterile pipette tip. Cells were washed with
PBS to remove dislodged cells. After adding fresh medium, cells were left in the incubator
for 24 more hours. Subsequently, micrographs were taken at three different positions and
analyzed with the Fiji ImageJ software (version 1.53e). The rate of migration was defined
as the percentage of wound closure area [27].
2.11. Real Time Cellular Analysis (RTCA)
The xCELLigence Real-Time Cell Analyzer
®
(Roche, Mannheim, Germany) was used
to measure growth characteristics such as proliferation rate and cell adhesion of VX2 cells,
in which the E6 gene was knocked down using siE6 RNA. The xCELLigence
®
technology
is based on impedance sensing where change in impedance is reported as a dimensionless
parameter called cell index (CI; CI = impedance at time point n
−
impedance without
cells/nominal impedance value). The magnitude of the CI depends upon the number
of cells, their size, and the degree of firmness of cell adhesion to the substrate coating
the plates [
28
]. Briefly, 96-well E-plates
®
(Roche), specifically designed for impedance
measurement of cells, were prepared by adding 150
µ
L of DMEM/Ham’s F-12 medium
containing serum and antibiotics into each well. Equilibration was achieved by placing the
plate into the xCELLigence station, and baseline electrical resistance was gauged to confirm
that all connections to the wells were working at appropriate limits. The E-plate was taken
out and 50
µ
L of untreated VX2 cell suspension was added to each well with a cell density
of 4.5
×
10
3
cells per well. Subsequently, an impedance measurement was performed over
a period of 24 h at 37
◦
C. The CI was calculated every 15 min. Once the VX2 cells began to
adhere to the support, the cell culture medium was removed and replaced with 175
µ
L of
fresh DMEM/Ham’s F-12 medium and 25
µ
L of an LPP solution loaded with 150 nmol/L
NT or siE6. The CI was measured in real time every 15 min for an additional 72 h.
2.12. Flow Cytometry and Cell Cycle Analysis of VX2 Cells
For cell cycle analysis, VX2 cells were transfected with LPPs loaded with siRNA
directed against the CRPV E6 and E7 oncogenes. Forty-eight hours after transfection,
cells were trypsinized and fixed for at least 2 h in ice-cold (
−
20
◦
C) 70% ethanol. After
centrifugation (300
×
g, 10 min) and removal of the ethanol, the pellet was resuspended
Pharmaceutics 2023,15, 2379 7 of 18
in 500
µ
L PBS, and RNAse A was added to a final concentration of 50
µ
g/mL followed
by an incubation for 4 h at 37
◦
C. Propidium iodide (PI; final concentration 5
µ
g/mL) was
added and cells were incubated for 10 min. Cell cycle analysis was performed on a BD
LSR II FACS analyzer (Becton Dickinson, Franklin Lakes, NJ, USA) by firstly deploying the
forward- (FSC) and side-scatter (SSC) parameters to define a single cell population and to
remove cell doublets. Measurement of fluorescence was performed in the PE-H channel.
Analysis was performed with the FlowJo
™
software (version 7.6.5, Tree Star Inc., Ashland,
OR, USA).
2.13. Statistical Analysis
All tests were performed using the Graph PadPrism software (version 9.0.0 for MAC;
GraphPad Software, Inc., San Diego, CA, USA). The following statistical analyses were
used: The one-tailed unpaired Students t-test for evaluating the basal expression of E6
and E7 mRNA, the cell viability and the E6/E7 mRNA expression, the FACS analysis of
GFP (geometric mean fluorescence) knockdown in COS-7 cells, transiently transfected
with GFP-E6- or GFP-E7-expressing plasmids, as well as the cell index measured by real-
time cellular analysis; the one-tailed unpaired Mann–Whitney assay for E6/E7 mRNA
expression after using different concentrations of siRNA; the two-tailed unpaired T-test with
Welch correction for the scratch assay; the two-tailed unpaired T-test for the distribution
of cells in each cell cycle phase. Data represent the mean
±
SD, with p< 0.05 considered
statistically significant. Statistical differences were indicated as *: p< 0.05, **: p< 0.01,
***: p< 0.001 and ****: p< 0.0001.
3. Results and Discussion
3.1. Generation of a VX2-Tumor-Derived Cell Line
VX2-tumor-derived tissue was cultured as described above. Early cell culture pas-
sages (passage #7) showed a mixture of vimentin (VIM)-positive and -negative cells
(Figure 1a). VIM is a typical marker for mesenchymal cells such as fibroblasts. Expectedly,
early passages should contain tumor-derived fibroblasts, which after repeated passages
should disappear due to senescence. Accordingly, after longer passages (passage #49), no
VIM-positive cells could be observed any more (Figure 1a). Earlier, we performed next-
generation sequencing (RNASeq) of VX2 tumor tissue-derived RNA [
29
]. In our present
study, we used these data to graphically depict which regions of the CRPV genome are
expressed in the VX2 tumor. A major expression is seen for the oncogenes E6 (SE6 and LE6),
E7, and E2, with the latter missing part of its coding sequence (Figure 1b). This expression
pattern is typically also found in other papillomavirus-associated cancers such as HPV-
driven human cancers [
30
]. Since other papillomavirus-associated genes, e.g., the late genes
L1 and L2, are virtually not expressed in these cancers, a major viral assembly and release
of complete viral particles from papillomavirus-driven cancer cells appears rather unlikely,
although this cannot be ruled out in some cases. To evaluate if the continuously growing,
morphologically homogenous, and VIM-negative cells are, indeed, VX2-tumor-derived, we
used RT-qPCR to monitor for expression of the major CRPV oncogenes E6 and E7. Both E6
and E7 viral transcripts were found to be significantly expressed in VX2-derived cultured
cells, which exhibited a tendency (not statistically significant) to lower expression levels
than in VX2 tumor tissues that were carried along as a positive control (Figure 1c). The
VX2-derived cell line survived for approximately 150 passages, which allowed for
in vitro
analysis of these cells in our study. Future studies aim to optimize culturing conditions to
obtain an indefinitely growing VX2 cell line. Interestingly, since many more HPV-negative
than HPV-positive cell lines exist than would be expected from the frequency of these
tumors, it appears likely that establishing HPV-positive HNSCC cell lines is less effective.
Pharmaceutics 2023,15, 2379 8 of 18
Pharmaceutics 2023, 15, x 8 of 19
tumor-derived, we used RT-qPCR to monitor for expression of the major CRPV oncogenes
E6 and E7. Both E6 and E7 viral transcripts were found to be significantly expressed in
VX2-derived cultured cells, which exhibited a tendency (not statistically significant) to
lower expression levels than in VX2 tumor tissues that were carried along as a positive
control (Figure 1c). The VX2-derived cell line survived for approximately 150 passages,
which allowed for in vitro analysis of these cells in our study. Future studies aim to opti-
mize culturing conditions to obtain an indefinitely growing VX2 cell line. Interestingly,
since many more HPV-negative than HPV-positive cell lines exist than would be expected
from the frequency of these tumors, it appears likely that establishing HPV-positive
HNSCC cell lines is less effective.
Figure 1. Generation of VX2 cell cultures. (a) Immunofluorescence analysis of VX2 cell cultures dur-
ing early (p #7) and late (p #49) passages demonstrating vimentin-expressing cells at early passages
only. (b) Pattern of gene expression of the CRPV genome. (c) CRPV E6 and E7 mRNA expression
levels in VX2-derived cell cultures compared with VX2 tissues. (E1, E2, E4, E5, LE6, SE6, E7, and E8
= “early” genes; L1 and L2 = “late” genes). Statistics: one-tailed unpaired Student’s t-test (n = 3 ex-
periments); significance level *: p < 0.05, n.s.: no significance.
3.2. Generation of Lipopolyplexes and Evaluation of Cellular Toxicity
Figure 1.
Generation of VX2 cell cultures. (
a
) Immunofluorescence analysis of VX2 cell cultures during
early (p #7) and late (p #49) passages demonstrating vimentin-expressing cells at early passages only.
(
b
) Pattern of gene expression of the CRPV genome. (
c
) CRPV E6 and E7 mRNA expression levels in
VX2-derived cell cultures compared with VX2 tissues. (E1, E2, E4, E5, LE6, SE6, E7, and E8 = “early”
genes; L1 and L2 = “late” genes). Statistics: one-tailed unpaired Student’s t-test (n = 3 experiments);
significance level *: p< 0.05, n.s.: no significance.
3.2. Generation of Lipopolyplexes and Evaluation of Cellular Toxicity
Lipopolyplexes (LPPs) (Figure 2a) were generated as described above. The major
limitation of most commercially available transfecting reagents is their high cytotoxicity [
31
].
The MTT assay was deployed to evaluate the effect on cell viability after exposing VX2
and COS-7 cells to LPPs. Cytotoxicity of LPPs was compared to Lipofectamine 2000
TM
(LF), a well-established
in vitro
transfection reagent. VX2 cells treated with LPPs exhibited
a tendency to higher viability, i.e., lower toxicity, compared to a treatment with LF. This
difference reached significance in COS-7 cells (Figure 2b,c). Overall, this points to a lower
toxicity of LPPs compared to LPs. These LPPs, therefore, could be promising candidates
for a deployment in vivo.
Pharmaceutics 2023,15, 2379 9 of 18
Pharmaceutics 2023, 15, x 9 of 19
Lipopolyplexes (LPPs) (Figure 2a) were generated as described above. The major lim-
itation of most commercially available transfecting reagents is their high cytotoxicity [31].
The MTT assay was deployed to evaluate the effect on cell viability after exposing VX2
and COS-7 cells to LPPs. Cytotoxicity of LPPs was compared to Lipofectamine 2000
TM
(LF), a well-established in vitro transfection reagent. VX2 cells treated with LPPs exhibited
a tendency to higher viability, i.e., lower toxicity, compared to a treatment with LF. This
difference reached significance in COS-7 cells (Figure 2b,c). Overall, this points to a lower
toxicity of LPPs compared to LPs. These LPPs, therefore, could be promising candidates
for a deployment in vivo.
Figure 2. Response of cells to transfection with lipopolyplexes (LPPs) in comparison with Lipofec-
tamine 2000
TM
(LF). (a) Schematic, depicting assembly of LPPs. (b,c) Effect on viability of (b) VX2
and (c) COS-7 cells after transfection with LF or LPPs. (d,e) Level of CRPV E7 mRNA expression
after transfecting (d) VX2 and (e) COS-7 cells with CRPV E7-expressing plasmids using LF or LPPs.
Statistics: one-tailed unpaired Student’s t-test (n = 3 experiments); significance level *: p < 0.05, **: p
< 0.01, and ****: p < 0.0001, n.s.: no significance.
Figure 2.
Response of cells to transfection with lipopolyplexes (LPPs) in comparison with Lipofec-
tamine 2000
TM
(LF). (
a
) Schematic, depicting assembly of LPPs. (
b
,
c
) Effect on viability of (
b
) VX2
and (
c
) COS-7 cells after transfection with LF or LPPs. (
d
,
e
) Level of CRPV E7 mRNA expression
after transfecting (
d
) VX2 and (
e
) COS-7 cells with CRPV E7-expressing plasmids using LF or LPPs.
Statistics: one-tailed unpaired Student’s t-test (n = 3 experiments); significance level *: p< 0.05,
**: p< 0.01, and ****: p< 0.0001, n.s.: no significance.
3.3. Transfection Efficiency of Lipopolyplexes in VX2 and COS-7 Cells
In order to quantify the transfection efficiency of LPPs, VX2 and COS-7 cells were
transfected with CRPV-E7-expressing plasmids. Expression of transcripts was evaluated
by RT-qPCR, as shown in Figure 2d,e. Untransfected VX2 and COS-7 cells served as a
negative control, whereas cells transfected with LF were used as positive control. CRPV
E7 transcripts were detected in highly significant amounts in VX2 as well as COS-7 cells.
Expression of E7 transcripts was higher in VX2 cells after transfection with LF, whereas no
difference in performance between LPPs and LF was seen in COS-7 cells (Figure 2d,e).
Pharmaceutics 2023,15, 2379 10 of 18
3.4. Expression of GFP- and RFP-Tagged CRPV-E6 and -E7 Oncoproteins in VX2 and
COS-7 Cells
One drawback when working with CRPV E6 and E7 is the lack of suitable antibodies
for detection of these viral oncoproteins. Against this background, we generated chimeric
GFP- and RFP-tagged CRPV-E6 and -E7 expression plasmids, which should enable the
monitoring of biological effects on E6 and E7 expression
in vitro
using fluorescence mi-
croscopy and flow cytometry. Transient transfection of GFP- and RFP-tagged CRPV-E6-
and -E7-expressing plasmids demonstrated pronounced expression levels in COS-7 as well
as VX2 cells (Figure 3a,b). Antibodies directed against GFP or RFP could detect GFP-E7 and
RFP-E7 chimeric proteins during Western blot analysis in COS-7 and VX2 cells. However,
no expression of GFP-E6 and RFP-E6 could be seen in our Western blot settings (Figure 3c).
Since GFP-E6 and -E7 were expressed in both COS-7 and VX2 cell lines, it appears likely
that the chimeric E6 protein becomes insoluble during sample preparation for Western blot
analysis. Interestingly, in a study by Liu et al., the authors described that HPV16-E6 under
specific conditions exhibits a tendency to become insoluble [
32
]. Similar structural features
of the CRPV-E6 protein could explain why the GFP/RFP-tagged CRPV-E6 protein does not
become soluble during cell lysis for Western blot analysis.
3.5. Effectiveness of CRPV E6 and E7 RNAi Knockdown Using siRNA-Loaded LPPs
Selecting suitable siRNA concentrations with significant knockdown potential yet
minimum cytotoxicity is a relevant first step. Here, several factors can influence siRNA-
mediated gene knockdown, such as choice of the siRNA sequence, concentration, trans-
fecting reagent, and the type of cell line. Next to VX2 cells, the CRPV-negative cell line
COS-7 was used as a VX2 cell independent test system for CRPV E6 and E7 RNAi studies
(Figure 4). For this, plasmids encoding CRPV_E6 and CRPV_E7 were transiently trans-
fected using low-toxicity LPPs as transfecting reagents. Here, the specificity and mRNA
knockdown efficiency of siRNAs directed against CRPV E6 and E7 oncogenes could be
assessed in a defined CRPV_E6- and CRPV_E7-expressing cell system (Figure 4a) and
compared to VX2 cells (Figure 4b). Efficient CRPV_E6 and CRPV_E7 mRNA knockdown
could be observed with 150 nmol/L siRNA in COS-7 cells (Figure 4a). However, VX2 cells
exhibited a rather paradoxical behavior, demonstrating a solid CRPV_E6 and CRPV_E7
mRNA knockdown at 50 nmol/L siRNA, whereas higher levels did not reach this efficiency
(Figure 4b). This observation could be explained by the fact that there is not always a
uniform concentration-dependent cellular uptake of siRNA/lipopolyplexes, which could
result in different knockdown efficiencies. Similarly, every N/P ratio of the polymer/siRNA
complex can result in a different knockdown efficiency which is not necessarily depen-
dent on the concentration of the siRNA. To evaluate the level of CRPV E6 and E7 protein
knockdown using siRNA-loaded LPPs, COS-7 cells expressing E6-GFP or E7-GFP were
treated with siRNA directed against E6 (siE6) or E7 (siE7). Flow cytometry analysis could
demonstrate a highly efficient knockdown of (GFP-)E6 and (GFP-)E7 measured by the
respective reduction in fluorescence intensity, which corresponded to the downmodulation
of E6 and E7 mRNA levels as seen during RT-qPCR analysis (Figure 4c). PCNA (prolif-
erating cell nuclear antigen) serves as a reliable proliferation marker that is responsible
for regulating the process of DNA replication during the S-phase of the cell cycle, thereby
having a vital role in cell proliferation. Western blot analysis was performed to evaluate the
effect of CRPV E6 and E7 RNAi knockdown on PCNA expression. Western blot analysis
of E6/E7-siRNA-treated cells showed a prominent decline in the PCNA protein levels,
which suggested a reduced proliferation. Protein bands were quantified based on optical
density/intensity using the ImageJ/Fiji software (version 1.54f) [24].
Pharmaceutics 2023,15, 2379 11 of 18
Pharmaceutics 2023, 15, x 11 of 19
Figure 3. Expression of CRPV oncoproteins E6 and E7 in COS-7 and VX2 cells. Fluorescence micro-
scopic images of COS-7 (a) and VX2 (b) cells transfected with viral oncoprotein-expressing plasmids
tagged with GFP or RFP. GFP_E6 or GFP_E7 expression (arrows) is depicted in the green (GFP)
channel, whereas RFP_E6 or RFP_E7 expression (arrows) is seen in the red (RFP) channel. Nontrans-
fected cells (blank) were used for background labeling. (c) Western blot analysis, demonstrating
expression of GFP-E7 and RFP-E7 recombinant proteins of expected size in transfected COS-7 and
VX2 cells. No specific signal is seen for GFP-E6 and RFP-E6.
3.5. Effectiveness of CRPV E6 and E7 RNAi Knockdown Using siRNA-Loaded LPPs
Selecting suitable siRNA concentrations with significant knockdown potential yet
minimum cytotoxicity is a relevant first step. Here, several factors can influence siRNA-
mediated gene knockdown, such as choice of the siRNA sequence, concentration, trans-
fecting reagent, and the type of cell line. Next to VX2 cells, the CRPV-negative cell line
COS-7 was used as a VX2 cell independent test system for CRPV E6 and E7 RNAi studies
(Figure 4). For this, plasmids encoding CRPV_E6 and CRPV_E7 were transiently trans-
fected using low-toxicity LPPs as transfecting reagents. Here, the specificity and mRNA
knockdown efficiency of siRNAs directed against CRPV E6 and E7 oncogenes could be
assessed in a defined CRPV_E6- and CRPV_E7-expressing cell system (Figure 4a) and
compared to VX2 cells (Figure 4b). Efficient CRPV_E6 and CRPV_E7 mRNA knockdown
could be observed with 150 nmol/L siRNA in COS-7 cells (Figure 4a). However, VX2 cells
Figure 3.
Expression of CRPV oncoproteins E6 and E7 in COS-7 and VX2 cells. Fluorescence micro-
scopic images of COS-7 (
a
) and VX2 (
b
) cells transfected with viral oncoprotein-expressing plasmids
tagged with GFP or RFP. GFP_E6 or GFP_E7 expression (arrows) is depicted in the green (GFP) chan-
nel, whereas RFP_E6 or RFP_E7 expression (arrows) is seen in the red (RFP) channel. Nontransfected
cells (blank) were used for background labeling. (
c
) Western blot analysis, demonstrating expression
of GFP-E7 and RFP-E7 recombinant proteins of expected size in transfected COS-7 and VX2 cells. No
specific signal is seen for GFP-E6 and RFP-E6.
3.6. Wound Closure Assay and Real-Time Cellular Analysis (RTCA)
Previously, it was found that knockdown of E6 and E7 alone or in combination elicits
an effect on the rate of cell migration
in vitro
in the HPV-16-positive cervical cancer cell
lines Caski and SiHa [
33
]. To evaluate the influence of CRPV E6 and E7 on cell migration in
VX2 cells, we performed a wound healing assay. The rate of migration was defined as the
percentage of wound (scratch) closure area. Analysis revealed that 24 h after applying a
defined scratch in a confluent VX2 cell layer, closure of the wounded area was significantly
lower after RNAi knockdown of E6/E7 (Figure 5a). The data suggest that downregulation
of the CRPV oncogenes E6 and E7, individually or combined, inhibits cell migration of
VX2 cells. To test whether this effect could be caused by a decreased growth rate after
E6/E7 knockdown, the growth curves of VX2 cells with and without knockdown of E6
Pharmaceutics 2023,15, 2379 12 of 18
were measured in a real-time cellular analysis assay (RTCA) (Figure 5b). This RTCA system
(xCELLigence) can be used to monitor live cell viability, proliferation, motility, adhesion,
migration, invasion, cell number, and morphology. It provides label-free and real-time
surveillance of cell viability by tracking the electrical impedance as a readout [
34
]. The
data show a decreased proliferation rate for the entire duration of the electrical impedance
recording (72 h) and a significantly decreased proliferation rate after downregulation of E6,
indicating an overall decrease in growth rate.
Pharmaceutics 2023, 15, x 12 of 19
exhibited a rather paradoxical behavior, demonstrating a solid CRPV_E6 and CRPV_E7
mRNA knockdown at 50 nmol/L siRNA, whereas higher levels did not reach this effi-
ciency (Figure 4b). This observation could be explained by the fact that there is not always
a uniform concentration-dependent cellular uptake of siRNA/lipopolyplexes, which
could result in different knockdown efficiencies. Similarly, every N/P ratio of the poly-
mer/siRNA complex can result in a different knockdown efficiency which is not neces-
sarily dependent on the concentration of the siRNA. To evaluate the level of CRPV E6 and
E7 protein knockdown using siRNA-loaded LPPs, COS-7 cells expressing E6-GFP or E7-
GFP were treated with siRNA directed against E6 (siE6) or E7 (siE7). Flow cytometry anal-
ysis could demonstrate a highly efficient knockdown of (GFP-)E6 and (GFP-)E7 measured
by the respective reduction in fluorescence intensity, which corresponded to the
downmodulation of E6 and E7 mRNA levels as seen during RT-qPCR analysis (Figure 4c).
PCNA (proliferating cell nuclear antigen) serves as a reliable proliferation marker that is
responsible for regulating the process of DNA replication during the S-phase of the cell
cycle, thereby having a vital role in cell proliferation. Western blot analysis was performed
to evaluate the effect of CRPV E6 and E7 RNAi knockdown on PCNA expression. Western
blot analysis of E6/E7-siRNA-treated cells showed a prominent decline in the PCNA pro-
tein levels, which suggested a reduced proliferation. Protein bands were quantified based
on optical density/intensity using the ImageJ/Fiji software (version 1.54f) [24].
Figure 4. RNAi knockdown of CRPV E6 and E7 using siRNA-loaded LPPs. Effect of CRPV E6 and
E7 RNAi knockdown in COS-7 (a) and VX2 (b) cells transfected with E6- and E7-expressing plas-
mids, using different siRNA concentrations (50, 100, and 150 nmol/L), on the mRNA expression
levels of these oncogenes. (c) Prominent downregulation of the GFP signal (geometric mean fluo-
rescence) after E6 and E7 RNAi in COS-7 cells, transiently transfected with GFP-E6- or GFP-E7-
expressing plasmids. (d) RNAi knockdown of CRPV E6 and E7 results in reduction of PCNA ex-
pression. Statistics: (a,b) one-tailed unpaired Mann–Whitney assay; (c) one-tailed unpaired Stu-
dent’s t-test (n = 3 experiments); significance level *: p < 0.05, **: p < 0.01, ***: p < 0.001, and ****: p <
0.0001.
3.6. Wound Closure Assay and Real-Time Cellular Analysis (RTCA)
Figure 4.
RNAi knockdown of CRPV E6 and E7 using siRNA-loaded LPPs. Effect of CRPV E6 and E7
RNAi knockdown in COS-7 (
a
) and VX2 (
b
) cells transfected with E6- and E7-expressing plasmids,
using different siRNA concentrations (50, 100, and 150 nmol/L), on the mRNA expression levels of
these oncogenes. (
c
) Prominent downregulation of the GFP signal (geometric mean fluorescence) after
E6 and E7 RNAi in COS-7 cells, transiently transfected with GFP-E6- or GFP-E7-expressing plasmids.
(
d
) RNAi knockdown of CRPV E6 and E7 results in reduction of PCNA expression. Statistics: (
a
,
b
)
one-tailed unpaired Mann–Whitney assay; (
c
) one-tailed unpaired Student’s t-test (n = 3 experiments);
significance level *: p< 0.05, **: p< 0.01, ***: p< 0.001, and ****: p< 0.0001.
Pharmaceutics 2023,15, 2379 13 of 18
Pharmaceutics 2023, 15, x 14 of 19
Figure 5. Functional effects of E6/E7 knockdown in VX2 cells on their migratory ability and prolif-
eration rate. VX2 cells were transfected by LPP-carrying nontarget (NT) RNA, siE6 RNA, siE7 RNA,
or siE6 + siE7 RNA in combination. (a) Representative microscopic images of wound healing assays
using VX2 cells are shown. The baseline situation of the wound healing assay at time 0, immediately
after the scratch, is shown as an example for the NT siRNA. All other microscopic images show the
in vitro cultures 24 h after the scratch. The blue line marks the boundary between open and closed
scratch; the yellow line represents the length of the scratch used for the statistical analysis of scratch
(wound) closure, as shown in the associated graph. All wound healing experiments were performed
in triplicates. (b) Real-time cellular analysis of the proliferation rate of VX2 cells after CRPV E6
knockdown. VX2 cells were treated with LPPs, loaded with 50 nmol/L NT control RNA (b1) or siE6
RNA (b2). Each colored line in the top 2 graphs corresponds to a measurement in the xCELLigence
Real-Time Cell Analyzer®. The lower left graph (b3) shows the mean values from each of the six
independent measurements, the lower right graph (b4) depicts the determined proliferation rate.
Mean +/− SD are shown in (a). Statistical differences of the used two-tailed unpaired t-test with
Welch correction for (a) and the one-tailed unpaired Student’s t-test for (b) were indicated as *: p <
0.05, **: p < 0.01.
Figure 5.
Functional effects of E6/E7 knockdown in VX2 cells on their migratory ability and prolifer-
ation rate. VX2 cells were transfected by LPP-carrying nontarget (NT) RNA, siE6 RNA, siE7 RNA, or
siE6 + siE7 RNA in combination. (
a
) Representative microscopic images of wound healing assays
using VX2 cells are shown. The baseline situation of the wound healing assay at time 0, immediately
after the scratch, is shown as an example for the NT siRNA. All other microscopic images show
the
in vitro
cultures 24 h after the scratch. The blue line marks the boundary between open and
closed scratch; the yellow line represents the length of the scratch used for the statistical analysis of
scratch (wound) closure, as shown in the associated graph. All wound healing experiments were
performed in triplicates. (
b
) Real-time cellular analysis of the proliferation rate of VX2 cells after
CRPV E6 knockdown. VX2 cells were treated with LPPs, loaded with 50 nmol/L NT control RNA
(b1) or siE6 RNA (b2). Each colored line in the top 2 graphs corresponds to a measurement in the
xCELLigence Real-Time Cell Analyzer
®
. The lower left graph (b3) shows the mean values from each
of the six independent measurements, the lower right graph (b4) depicts the determined proliferation
rate. Mean +/
−
SD are shown in (
a
). Statistical differences of the used two-tailed unpaired t-test
with Welch correction for (
a
) and the one-tailed unpaired Student’s t-test for (
b
) were indicated as
*: p< 0.05, **: p< 0.01.
Pharmaceutics 2023,15, 2379 14 of 18
3.7. Indication of G1 Block after CRPV E6 and E7 Knockdown in VX2 Cells
The effect of CRPV oncogene RNAi knockdown on the cell cycle of cultured VX2 cells
was evaluated by flow cytometry. Consistent with the literature on long-term VX2 cell
cultures [
35
], in untreated VX2 cells, 50.2% (+/
−
2.0%) of cells were in the G0/G1 resting
phase, 32.2% (+/
−
1.2%) were in the S replication phase, and 17.2% (+/
−
0.4%) were in the
G2/M growth phase (Figure 6a,b). Elimination of CRPV E6 or E7 alone or in combination
resulted in a significant arrest of VX2 cells in the G0/G1 phase of the cell cycle compared
to the control (NT), which was accompanied by a reduced number of cells in the S and
G2/M phases of the cell cycle, which in some cases reached significance (Figure 6b). The
cell cycle analysis data correlate with cell viability, which decreased significantly in all
three knockdown groups (siE6, siE7, and siE6 + siE7) (Figure 6c), suggesting that E6 and
E7 support tumorigenic growth of VX2 cells by promoting entry into the mitotic phase,
which is a well-known feature for these oncoproteins [
36
]. Similarly, previous studies
demonstrated a role of the CRPV E6 protein in protecting tumor cells from apoptosis and
maintaining cancer cell population integrity [
37
]. These reports are in agreement with our
observations in VX2 cells. Furthermore, various studies have previously demonstrated
that CRPV E6 and E7 oncogenes play a vital role in the transformation and anchorage-
independent growth of tumor cells [
38
]. It can, therefore, be expected that RNAi-mediated
downregulation of these genes affects the rate of VX2 cell proliferation.
Pharmaceutics 2023, 15, x 15 of 19
3.7. Indication of G1 Block after CRPV E6 and E7 Knockdown in VX2 Cells
The effect of CRPV oncogene RNAi knockdown on the cell cycle of cultured VX2 cells
was evaluated by flow cytometry. Consistent with the literature on long-term VX2 cell
cultures [35], in untreated VX2 cells, 50.2% (+/−2.0%) of cells were in the G0/G1 resting
phase, 32.2% (+/−1.2%) were in the S replication phase, and 17.2% (+/−0.4%) were in the
G2/M growth phase (Figure 6a,b). Elimination of CRPV E6 or E7 alone or in combination
resulted in a significant arrest of VX2 cells in the G0/G1 phase of the cell cycle compared
to the control (NT), which was accompanied by a reduced number of cells in the S and
G2/M phases of the cell cycle, which in some cases reached significance (Figure 6b). The
cell cycle analysis data correlate with cell viability, which decreased significantly in all
three knockdown groups (siE6, siE7, and siE6 + siE7) (Figure 6c), suggesting that E6 and
E7 support tumorigenic growth of VX2 cells by promoting entry into the mitotic phase,
which is a well-known feature for these oncoproteins [36]. Similarly, previous studies
demonstrated a role of the CRPV E6 protein in protecting tumor cells from apoptosis and
maintaining cancer cell population integrity [37]. These reports are in agreement with our
observations in VX2 cells. Furthermore, various studies have previously demonstrated
that CRPV E6 and E7 oncogenes play a vital role in the transformation and anchorage-
independent growth of tumor cells [38]. It can, therefore, be expected that RNAi-mediated
downregulation of these genes affects the rate of VX2 cell proliferation.
Figure 6. Effect of E6/E7 knockdown on the cell cycle and viability of VX2 cells. (a) Representative
cell cycle histograms of VX2 cells after RNAi knockdown of the CRPV oncogenes E6 and E7. (b)
Percentage of VX2 cells in each cell cycle phase according to the respective treatment group. (c)
Effect on VX2 cell viability after treatment with siE6-, siE7-, or siE6 + siE7-loaded LPPs (“non” indi-
cates the group of untreated VX2 cells; “NT” indicates the group of VX2 cells treated with nontarget-
RNA-loaded LPPs). All experiments were performed in triplicate. Means +/− SD are shown. Statis-
tical differences were evaluated with the unpaired two-tailed Student’s t-test and (b) the unpaired
one-tailed Student’s t-test (c), and are indicated as *: p < 0.05, **: p < 0.01, and ***: p < 0.001, n.s.: no
significance.
Previous reports indicated roles of CRPV E6 and E7 as key genes for papilloma for-
mation in rabbits and also addressed the importance of these genes as possible therapeutic
targets for CRPV-induced carcinomas [29]. Downregulation or suppression of either of
these genes may, therefore, cause a significant decline in papilloma development. Another
Figure 6.
Effect of E6/E7 knockdown on the cell cycle and viability of VX2 cells. (
a
) Representa-
tive cell cycle histograms of VX2 cells after RNAi knockdown of the CRPV oncogenes E6 and E7.
(
b
) Percentage of VX2 cells in each cell cycle phase according to the respective treatment group.
(
c
) Effect on VX2 cell viability after treatment with siE6-, siE7-, or siE6 + siE7-loaded LPPs (“non”
indicates the group of untreated VX2 cells; “NT” indicates the group of VX2 cells treated with
nontarget-RNA-loaded LPPs). All experiments were performed in triplicate. Means +/
−
SD are
shown. Statistical differences were evaluated with the unpaired two-tailed Student’s t-test and
(
b
) the unpaired one-tailed Student’s t-test (
c
), and are indicated as *: p< 0.05, **: p< 0.01, and
***: p< 0.001, n.s.: no significance.
Previous reports indicated roles of CRPV E6 and E7 as key genes for papilloma forma-
tion in rabbits and also addressed the importance of these genes as possible therapeutic
targets for CRPV-induced carcinomas [
29
]. Downregulation or suppression of either of
these genes may, therefore, cause a significant decline in papilloma development. Another
Pharmaceutics 2023,15, 2379 15 of 18
study reported the outcome of CRPV E1, E2, E6, and E7 vaccination, both individually
and in combination, in rabbits previously injected with CRPV viral DNA at specific sites.
Papillomas grew in all (100%) nonvaccinated rabbits. Rabbits vaccinated with a single-
gene vaccine were only partially protected against papilloma growth on the challenged
sites. However, two out of four rabbits vaccinated with an E1, E2, E6, and E7 combination
vaccine became completely free of papillomas, while in the other two rabbits only small
papillomas developed at the challenged sites, which regressed within three weeks. This
outcome showed that CRPV oncoproteins, when targeted in combination, may lead to
synergistic therapeutic effects [
24
]. These findings are in agreement with those of our study,
which demonstrate that RNAi-mediated knockdown of CRPV E6 and E7 results in re-
duced viability, migration, and proliferation by inducing a block in the G0/G1 phase of the
cell cycle.
In addition to toxicity, the selectivity of gene transfection systems is decisive for their
applicability as therapeutic agents in the patient. The lipopolyplexes we use are a further
development of the classic polyplexes using the optimum biocompatibility of the lipoplexes.
It has been known for many years that polyplexes have a very good transfection efficiency
in vitro
(e.g., cell culture), but many problems in the living organism include, among other
things, incompatibility with blood, poor trafficking to the target site, and accumulation in
the liver or lungs. The lipid envelope present in lipopolyplexes should at least reduce such
problems. These lipopolyplexes are available as parenteral formulations and are preferably
designed for intravenous administration. Studies in animals observed the intravenous
injection route to be effective for tumor targeting [
24
,
39
]. Good biocompatibility and
sufficiently long circulation times enable accumulation in tumor tissues with an EPR
(enhanced permeability and retention) effect. The accumulation in the liver should also
be significantly reduced. Linder et al. and Kurosaki et al. were able to demonstrate
that lipopolyplexes showed a significantly improved transfection efficiency, with only
very low accumulation in the liver [
40
,
41
]. Similarly, Ali et al. were able to show in a
comparable drug delivery system that there was a reduction in liver accumulation after
lipid coating [42].
4. Conclusions
Our previous studies [
5
] demonstrated the suitability of the VX2 carcinoma as a
model system for HPV-associated HNSCC, since this tumor, similarly to HPV+ HNSCC, is
the consequence of a papillomavirus (cottontail rabbit papillomavirus, CRPV) infection.
Against this background, it was of utmost importance to implement a CRPV cell culture
model that allows us to perform studies
in vitro
. We demonstrated that transiently cultured
VX2 cells are a suitable cell culture model. Furthermore, an artificial CRPV model system,
based on transient transfection of CRPV E6 and E7 oncogenes into the well-established
cell line COS-7, allows us to perform controlled investigations regarding the effectiveness
of candidate therapeutics such as the E6/E7 siRNA-loaded LPPs. Since both cell systems
allow many of the essential investigations to be performed
in vitro
, they help in reducing
animal experiments according to the 3R rule (reduce, replace, refine). Lessons learned
from these studies could directly lead to the development of analogous therapeutics for the
treatment of HPV-positive head and neck tumors in human patients. In the present study,
we demonstrated the potential usefulness of E6/E7-siRNA-loaded LPPs as a treatment
option for papillomavirus-associated head and neck cancers. Notably, LPPs directed against
CRPV E6 and E7, as presented in our study, could be directly used as possible therapeutic
agents for diseases associated with CRPV such as rabbit skin papillomas and cancers. This
pharmaceutical LPP formulation is designed for parenteral administration (i.v., i.m., and
pulmonary). Evaluating the bioavailability and efficacy
in vivo
of these formulations will
be an interesting task for following studies.
Pharmaceutics 2023,15, 2379 16 of 18
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/pharmaceutics15102379/s1, Figure S1: Cloning of CRPV E6/E7
expression constructs; Figure S2: Sequence alignment of the E6_WT clone; Figure S3: Sequence
alignment of the E7_wt clone; Figure S4: Sequence alignment of the GFP_E6 clone; Figure S5: Sequence
alignment of the GFP_E7 clone; Figure S6: Sequence alignment of the RFP_E6 clone; Figure S7:
Sequence alignment of the RFP_E7 clone; Table S1: Oligo primers; Table S2: List of primer pairs
containing restriction sites for the generation of CRPV E6 and E7 expression plasmids; Table S3:
siRNA sequences.
Author Contributions:
Conceptualization, R.M., U.B. and M.B.; data curation, U.A., U.B., R.M. and
M.B.; formal analysis, U.A., S.R.P. and G.A.; funding acquisition, U.B. and B.A.S.; investigation, U.A.,
G.A., I.T., S.R.P., A.M., R.M. and M.B.; methodology, U.A., S.R.P., G.A., I.T., R.M. and U.B.; project
administration, U.B. and R.M.; resources, U.B. and R.M.; supervision, U.B. and R.M.; validation, U.A.,
S.R.P., I.T., M.B., U.B. and R.M.; writing—original draft, U.A., M.B., U.B. and R.M.; writing—review
and editing, U.A., M.B., S.R.P., B.A.S., I.T., U.B. and R.M. All authors have read and agreed to the
published version of the manuscript.
Funding:
U.A. and G.A. were supported by the German Academic Exchange Service (DAAD,
reference numbers: 91591603 (U.A.) and 91591623 (G.A.).
Institutional Review Board Statement:
The generation of a solid VX2 tumor tissue in a New Zealand
White (NZW) rabbit was conducted in accordance with international standards on animal welfare,
the European directive 2010/63/EU, and the German Animal Protection Law under a protocol
approved by the county administrative government authorities in Giessen, Germany (V54-19c 20
15 h 01 MR20/26 Nr. G42/2017, date of approval: 1 September 2017).
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Acknowledgments:
Technical assistance by Roswitha Peldszus and Maria Sadowski (Department of
Otorhinolaryngology, Head and Neck Surgery, University Hospital Marburg, Philipps-Universität
Marburg, Marburg, Germany) and Eva Mohr (Department of Pharmaceutics and Biopharmaceutics,
Philipps-Universität Marburg, Marburg, Germany) is profoundly appreciated. Support during flow
cytometry analysis by Gavin Giel (Flow Cytometry Core Facility, Faculty of Medicine, Philipps-
Universität Marburg, Director: C. Brendel) and during real-time cellular analysis by Pietro Di Fazio
(Department of Visceral Thoracic and Vascular Surgery, Philipps-Universität Marburg, Marburg,
Germany) is highly acknowledged. Data from this study are part of the doctoral theses of U.A. and
G.A.
Conflicts of Interest:
The authors declare no conflict of interest. The company had no role in
the design of the study; in the collection, analyses, or interpretation of data; in the writing of the
manuscript, and in the decision to publish the results.
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