CD40/CD40 ligand interactions and TNFα treatment reduce activity of P105 promoter of the human papilloma virus-18 in vitro
Background: Cervical carcinoma cells including those infected with the oncogenic human papilloma virus (HPV) and several cervical carcinoma cell lines show a strong expression of the CD40 receptor, unlike benign cervical epithelial cells infected with HPV. The functional relevance of this up-regulat...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Цитувати: | CD40/CD40 ligand interactions and TNFα treatment reduce activity of P105 promoter of the human papilloma virus-18 in vitro / A. Altenburg, M.B. Abdel-Naser, G. Nikolakis, T. Wild, N. Wojtalewicz // Experimental Oncology. — 2016 — Т. 38, № 1. — С. 22-25. — Бібліогр.: 24 назв. — англ. |
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irk-123456789-1379792018-06-18T03:03:41Z CD40/CD40 ligand interactions and TNFα treatment reduce activity of P105 promoter of the human papilloma virus-18 in vitro Altenburg, A. Abdel-Naser, M.B. Nikolakis, G. Wild, T. Wojtalewicz, N. Original contributions Background: Cervical carcinoma cells including those infected with the oncogenic human papilloma virus (HPV) and several cervical carcinoma cell lines show a strong expression of the CD40 receptor, unlike benign cervical epithelial cells infected with HPV. The functional relevance of this up-regulated expression in the tumor is not fully understood. Nevertheless, it might offer a unique possibility to target those malignant cells due to the antiviral and antitumoral effects of the CD40/CD40 ligand (CD40L) interactions. Aim: In vitro assessment of the effect of CD40L on HPV 18-P105 promoter activity and the subsequent release of IL-6 by the promoter transfected HeLaCD₄₀ cells, which express CD40 constitutively. Material and Methods: Transfection of HeLaCD₄₀ cells was achieved by electroporation after optimizing the parameters by the pCMV-β-Gal vector and β-Gal stain. Transfected HeLaCD₄₀ cells were challenged with BHKCD40L and TNFα, in addition to BHKwt and medium alone as controls. HPV18P105 promoter activity was demonstrated by luciferase reporter gene assay while IL-6 was assessed by ELISA. Results: CD40/ CD40L interactions and TNFα treatment significantly reduced HPV18-P105 promoter activity (56.0 ± 10.2% and 64.1 ± 9.1% vs. control, respectively; p < 0.001). Likewise, IL-6, which is a sensitive cytokine of CD40 activation, was significantly increased in HeLaCD₄₀ cells in the same experiments (2.7 fold after stimulation with BHKCD₄₀L and 5.2 fold after stimulation with TNFα vs. control; p < 0.01 and p < 0.001, respectively). Conclusion: It is likely that the CD40/CD40L interactions and TNFα are effective against cervical carcinomas by repressing transcriptional activity of HPV promoter. This can result in new adjuvant treatments. 2016 Article CD40/CD40 ligand interactions and TNFα treatment reduce activity of P105 promoter of the human papilloma virus-18 in vitro / A. Altenburg, M.B. Abdel-Naser, G. Nikolakis, T. Wild, N. Wojtalewicz // Experimental Oncology. — 2016 — Т. 38, № 1. — С. 22-25. — Бібліогр.: 24 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/137979 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Altenburg, A. Abdel-Naser, M.B. Nikolakis, G. Wild, T. Wojtalewicz, N. CD40/CD40 ligand interactions and TNFα treatment reduce activity of P105 promoter of the human papilloma virus-18 in vitro Experimental Oncology |
| description |
Background: Cervical carcinoma cells including those infected with the oncogenic human papilloma virus (HPV) and several cervical carcinoma cell lines show a strong expression of the CD40 receptor, unlike benign cervical epithelial cells infected with HPV. The functional relevance of this up-regulated expression in the tumor is not fully understood. Nevertheless, it might offer a unique possibility to target those malignant cells due to the antiviral and antitumoral effects of the CD40/CD40 ligand (CD40L) interactions. Aim: In vitro assessment of the effect of CD40L on HPV 18-P105 promoter activity and the subsequent release of IL-6 by the promoter transfected HeLaCD₄₀ cells, which express CD40 constitutively. Material and Methods: Transfection of HeLaCD₄₀ cells was achieved by electroporation after optimizing the parameters by the pCMV-β-Gal vector and β-Gal stain. Transfected HeLaCD₄₀ cells were challenged with BHKCD40L and TNFα, in addition to BHKwt and medium alone as controls. HPV18P105 promoter activity was demonstrated by luciferase reporter gene assay while IL-6 was assessed by ELISA. Results: CD40/ CD40L interactions and TNFα treatment significantly reduced HPV18-P105 promoter activity (56.0 ± 10.2% and 64.1 ± 9.1% vs. control, respectively; p < 0.001). Likewise, IL-6, which is a sensitive cytokine of CD40 activation, was significantly increased in HeLaCD₄₀ cells in the same experiments (2.7 fold after stimulation with BHKCD₄₀L and 5.2 fold after stimulation with TNFα vs. control; p < 0.01 and p < 0.001, respectively). Conclusion: It is likely that the CD40/CD40L interactions and TNFα are effective against cervical carcinomas by repressing transcriptional activity of HPV promoter. This can result in new adjuvant treatments. |
| format |
Article |
| author |
Altenburg, A. Abdel-Naser, M.B. Nikolakis, G. Wild, T. Wojtalewicz, N. |
| author_facet |
Altenburg, A. Abdel-Naser, M.B. Nikolakis, G. Wild, T. Wojtalewicz, N. |
| author_sort |
Altenburg, A. |
| title |
CD40/CD40 ligand interactions and TNFα treatment reduce activity of P105 promoter of the human papilloma virus-18 in vitro |
| title_short |
CD40/CD40 ligand interactions and TNFα treatment reduce activity of P105 promoter of the human papilloma virus-18 in vitro |
| title_full |
CD40/CD40 ligand interactions and TNFα treatment reduce activity of P105 promoter of the human papilloma virus-18 in vitro |
| title_fullStr |
CD40/CD40 ligand interactions and TNFα treatment reduce activity of P105 promoter of the human papilloma virus-18 in vitro |
| title_full_unstemmed |
CD40/CD40 ligand interactions and TNFα treatment reduce activity of P105 promoter of the human papilloma virus-18 in vitro |
| title_sort |
cd40/cd40 ligand interactions and tnfα treatment reduce activity of p105 promoter of the human papilloma virus-18 in vitro |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2016 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/137979 |
| citation_txt |
CD40/CD40 ligand interactions and TNFα treatment reduce activity of P105 promoter of the human papilloma virus-18 in vitro / A. Altenburg, M.B. Abdel-Naser, G. Nikolakis, T. Wild, N. Wojtalewicz // Experimental Oncology. — 2016 — Т. 38, № 1. — С. 22-25. — Бібліогр.: 24 назв. — англ. |
| series |
Experimental Oncology |
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2025-07-10T02:41:40Z |
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2025-07-10T02:41:40Z |
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| fulltext |
22 Experimental Oncology 38, 22–25, 2016 (March)
CD40/CD40 LIGAND INTERACTIONS AND TNFα TREATMENT
REDUCE ACTIVITY OF P105 PROMOTER OF THE HUMAN
PAPILLOMA VIRUS-18 IN VITRO
A. Altenburg1,*, M.B. Abdel-Naser1,2, G. Nikolakis1, T. Wild1, N. Wojtalewicz1,3
1Department of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center,
Dessau D-06847, Germany
2Department of Dermatology, Ain Shams University, Cairo 11566, Egypt
³Center of Life Sciences Institute of Bioanalytical Sciences (IBAS), Anhalt University of Applied Sciences,
Bernburg D-06406, Germany
Background: Cervical carcinoma cells including those infected with the oncogenic human papilloma virus (HPV) and several cervi-
cal carcinoma cell lines show a strong expression of the CD40 receptor, unlike benign cervical epithelial cells infected with
HPV. The functional relevance of this up-regulated expression in the tumor is not fully understood. Nevertheless, it might offer
a unique possibility to target those malignant cells due to the antiviral and antitumoral effects of the CD40/CD40 ligand (CD40L)
interactions. Aim: In vitro assessment of the effect of CD40L on HPV 18-P105 promoter activity and the subsequent release
of IL-6 by the promoter transfected HeLaCD40 cells, which express CD40 constitutively. Material and Methods: Transfection
of HeLaCD40 cells was achieved by electroporation after optimizing the parameters by the pCMV-β-Gal vector and β-Gal stain.
Transfected HeLaCD40 cells were challenged with BHKCD40L and TNFα, in addition to BHKwt and medium alone as controls. HPV18-
P105 promoter activity was demonstrated by luciferase reporter gene assay while IL-6 was assessed by ELISA. Results: CD40/
CD40L interactions and TNFα treatment significantly reduced HPV18-P105 promoter activity (56.0 ± 10.2% and 64.1 ± 9.1%
vs. control, respectively; p < 0.001). Likewise, IL-6, which is a sensitive cytokine of CD40 activation, was significantly increased
in HeLaCD40 cells in the same experiments (2.7 fold after stimulation with BHKCD40L and 5.2 fold after stimulation with TNFα
vs. control; p < 0.01 and p < 0.001, respectively). Conclusion: It is likely that the CD40/CD40L interactions and TNFα are effec-
tive against cervical carcinomas by repressing transcriptional activity of HPV promoter. This can result in new adjuvant treatments.
Key Words: human papilloma virus, CD40, CD40 ligand, promoter, repression.
INTRODUCTION
CD40 is a member of the TNFR family. It is ex-
pressed on B cells, monocytes, dendritic cells, and
a variety of nonhemopoietic cells, including normal ke-
ratinocytes, tumor cells, and many in vitro transformed
and carcinoma-derived cell lines. CD40L, a member
of the TNF superfamily, is expressed primarily by acti-
vated T cells, activated B cells and platelets and under
inflammatory conditions on monocytic cells, natural
killer cells and mast cells. CD40/CD40L engagement
is required for B-cell differentiation, isotype switching,
maturation of dendritic cells and subsequent stimula-
tion of antigen-specific T cells [1].
The CD40/CD40L interactions in tumor cells seem
to be pleiotropic and context dependent as it may not
only inhibit tumor growth via immunologic mechanisms
and induction of apoptosis but also may stimulate tu-
mor growth via various cytokines and growth factors
including IL-6 and VEGF, among others [2]. Likewise,
TNFα exerts selective direct tumor cytotoxicity but
also promotes tumor development and metastases
via NO release and angiogenesis [3]. In vivo and
in vitro studies revealed that oncogenic human papil-
loma viruses (HPV) positive cervical carcinoma and
HPV positive cervical carcinoma cell lines expressed
CD40 at the high levels, in contrast to normal cervical
epithelium and non-malignant keratinocyte cell lines
transfected with HPV-DNA [4, 5]. This discrepancy
may provide an explanation for persistence of HPV
infection by evading the immune system as shown
by increased production of IL-18 binding protein that
antagonizes the inflammatory cytokine IL-18 and at-
tenuates the response to CD40 ligation and the capa-
city to attract peripheral blood mononuclear cells [6,
7]. This immune escape may be the rational for the
poor prognosis of cervical carcinoma in the presence
of HPV-18 genotype [8]. The selective effect of TNFα
on HPV infection is not known but previous studies
have shown that both CD40/CD40L interactions in the
presence of a protein inhibitor and TNFα exerted a di-
rect HeLaCD40 cytotoxicity, however it is unclear whether
this effect also affects HeLaCD40 cells’ HPV infection [9].
The aim of the present work is to examine the func-
tional consequences of CD40/CD40L interactions and
TNFα treatment on the HPV-18-p105 promoter activity
of transfected HeLaCD40 cells in vitro.
MATERIALS AND METHODS
Cell lines and formaldehyde fixation. The cervi-
cal carcinoma cell line HeLaCD40 (for cloning of CD40-
cDNA and transfection see [10], CCL 2; American
Submitted: May 26, 2015.
*Correspondence: E-mail: andreas.altenburg@klinikum-dessau.de
Abbreviations used: BHK — baby hamster kidney; CD40L —
CD40 ligand; HPV — human papilloma virus; IL-6 — interleukin-6;
IL-18 — interleukin-18; LCR — long control region; RLU — relative
light units; SN — supernatant; STAT3 — signal transducer and acti-
vator of transcription 3; TNF — tumor necrosis factor; VEGF — vas-
cular endothelial growth factor.
Exp Oncol 2016
38, 1, 22–25
Experimental Oncology 38, 22–25, 2016 (March) 23
Type Culture Collection, Kockville, MD) and the
baby hamster kidney (BHKCD40L) cells, transfected
with CD40L cDNA [11] (ACC 61; German Collection
of Micro-organisms and Cell Cultures, Braunschweig,
Germany) were cultured in DMEM supplemented with
10% heat-inactivated fetal calf serum, 1 mM sodium
pyruvate, 2 mM L-alanyl-L-glutamine and antibiotics
(all from Life Technologies, Eggenstein, Germany).
Formaldehyde-fixed BHKCD40L cells and the wild type
variant BHKwt were prepared as described previ-
ously [9]. Formaldehyde fixation of BHK cells prevents
cellular adhesion to culture plates and release of auto-
logous cytokines in an appreciable amount.
Transient transfection and assays. 1. Optimi-
zation of transfection efficiency by electroporation.
The pCMV-β-Gal vectors and β-Gal stain method
were used for adjustment and optimization of the
electroporation variable parameters as previously
described (Fig. 1, a) [12]. The voltage was varied
at a range of 220 to 350 V with stepwise incre-
ments of 10 V, while the capacitance was tested
at a range of 900 to 1400 µF with stepwise incre-
ments of 150 µF. The optimal parameters were found
to be 300 V/1050 µF as demonstrated by the count and
the density of the deep blue color of the transfected
cells (Fig. 1, b, c). 2. Reporter gene assays (Luciferase
assay). The luciferase expression test represents
a reporter system for analysis of regulatory DNA se-
quence in which luciferase catalyzes the D-luciferin
to emit a green luminescence that can be quantitatively
measured. Electroporation cuvettes, each containing
0.8 ml of HeLaCD40 cell suspension (adjusted at ca.
8–9•106 cells/ml), to which 10 µg 4321-luc DNA
(the entire HPV18 long control region (LCR) and the
p105 promoter are conjugated on the luciferase gene)
(Dr. Steger, Cologne, Germany) or 10 µg pCMV-β-Gal
vector that served as a control were added. Mixtures
were left at room temperature for 10 min and followed
by application of the optimum electric pulses (300 V,
1050 µF). Cells of 10 cuvettes were collected, gently
mixed and seeded at equal number in 20 culture plates
for 24 h followed by addition of formaldehyde-fixed
BHKCD40L (6 plates, 2•106 cells/plate), formaldehyde-
fixed BHKwt (6 plates, 2•106 cells/plate) and TNFα
(4 plates, each contains 1000 U/ml) while the re-
maining 4 plates contained medium only and served
as controls. Culture plates were incubated at 37 °C for
43 h followed by a gentle wash with warm medium
to remove the BHK cells and TNFα. The transfected
cells were washed with cold PBS and retrieved
by a rubber scraper in 1.8 ml PBS. After centrifugation
(250 g, 4 °C, 7 min) cells were resuspended in 50 µl ex-
traction buffer, lysed by 4 freeze-thaw cycles (each for
2 min in liquid nitrogen at −196 °C and 2 min in water
bath at 37 °C), and followed by ultracentrifugation
(1400 g, 5 min at 4 °C). The extract was retrieved and
kept on ice. The luciferase activity was measured
by mixing 15 µl of the extract with 100 µl of the lucif-
erase assay buffer (containing rATP). After injection
of 300 µl solvent (0.356 mM D-luciferin dissolved
in luciferase buffer) the relative light units (RLU) were
immediately measured in a LB 9501 luminometer
(Berthold Technologies, Bad Wildbad, Germany).
On the other hand, the control reaction consisted
of 470 µl β-galactosidase-buffer, 100 µl ONPG so-
lution (4 mg/ml O-Nitrophenol-β-D-galactosidase
in 0.1 M Na2HPO4-buffer, pH 7) and 30 µl cell extract
which were incubated till they attain yellow color. Wells
with β-Gal buffer and ONPG only served as controls.
After stopping the reaction with 250 µl NaCO2, the
fluorescence was measured at a wave length of 420 nm
(Fig. 1, a).
HeLaCD40
Optimization of transfection by electroporation with the plasmid
pCMV-βGal (β-Gal stain)
Electroporation with the reporter plasmids p4321-luc and pCMV-βGal
Incubation (43 h)
*Medium *†BHKCD40L *†BHKwt *TNFα
Assessment of promoter
activity by luciferase* and
β-galactosidase-assays†
*Assessment
of IL-6 of super-
natants (ELISA)
b
a
c
Fig. 1. Schematic representation of the experimental design,
steps and methods (a). Detection of β-galactosidase activity
after electrotransfection of HeLaCD40 cells (with pCMV-β-Gal;
original magnification, × 40; b — electroporation parameters
240 V and 1050 µF; c — electroporation parameters 300 V and
1050 µF; the latter represents the optimal parameters as shown
by the count and density of the blue dye)
Assessment of IL-6 by ELISA. Cells were seeded
in 24-well plates at a density of 1.5•105 cells/well and
were challenged after 24 h with BHKCD40L cells, BHKwt
cells (each with 2•106 BHK/well), TNFα (1000 U/ml)
or left with medium alone that served as a control.
After 16 h, supernatants (SNs) were collected, cel-
lular debris was removed by centrifugation and the
IL-6 contents were assessed by ELISA as previously
described [11]. Briefly, Maxisorp plates (Nunc, Wi-
esbaden, Germany) were coated with 1 mg/ml anti-
IL-6 overnight. After blo cking of the plates for 1 h with
PBS containing 0.5% BSA, 0.05% Tween 20 (Serva,
Heidelberg, Germany), and 0.02% NaN3, SNs and
serial dilutions of the recombinant human IL-6 (Tebu,
Frankfurt, Germany) as standards were added for
6 h. Plates were then incubated with polyclonal anti-
24 Experimental Oncology 38, 22–25, 2016 (March)
IL-6 overnight (Tebu, Frankfurt, Germany) followed
by a 2-hour incubation with peroxidase-labeled goat
anti-rabbit F(ab) after which the substrate was applied
and the absorbance was measured with an ELISA
reader (SLT Lab Instruments, Hannover) at 405 nm
(Fig. 1, a).
Statistical analysis. The mean and standard
deviation of values obtained from each assay were
calculated and the percentage decrease of luciferase
assays was estimated. Unpaired Student t test was
used to evaluate the difference between means and
SD of reporter gene assay and IL-6.
RESULTS
CD40/CD40L interactions and TNFα treatment
reduced the HPV18-P105-promoter activity in HeLa
cells that expressed CD40 constitutively. To exa-
mine the possible regulatory effect of CD40L on the
HPV18 control region, the HeLaCD40 cells transfected
with reporter gene were stimulated for 43 h with
formaldehyde-fixed BHKCD40L cells and also with
TNFα. Formaldehyde-fixed BHKwt and medium alone
served as controls. The activity of HPV18-P105-
promoter stimulated by formaldehyde-fixed BHKCD40L
(RLU 2902.3 ± 291.49 vs. 4488 ± 459.62; p = 0.017)
(in the four experiments) was repressed by a mean
of 56 ± 10.2%, in comparison with BHKwt (RLU 5114 ±
917.32 vs. 4488 ± 459.62; p = 0.45). Similarly, they
were significantly stimulated by 1000 U/ml TNFα (RLU
1765 ± 321.02 vs. 4488 ± 459.62; p = 0.02). On the
other hand, formaldehyde-fixed BHKwt induced no sig-
nificant effect in comparison with control medium (RLU
5114 ± 917.32 vs. 4488 ± 459.62; p = 0.45) (Fig. 2, a).
It has been shown earlier that TNFα negatively regu-
lates the HPV16 gene expression [13]. In the pre sent
work, activity of the HPV18-P105-promoter was
repressed by TNFα by a mean of 64.9 ± 9.1% in com-
parison with medium. The standard deviations from
several probes within each experiment were between
1.0 and 10.1%. In contrast, the control pCMV-β-Gal
vector-transfected HeLaCD40 cells incubated with
formaldehyde-fixed BHKCD40L or BHKwt cells or TNFα
or medium alone showed similar and non-significant
CMV promoter activity in all probes (p > 0.05).
CD40/CD40L interactions and TNFα treatment
induce IL-6 release by the HeLaCD40 cells transfected
with HPV18-P105. As IL-6 represents a very sensi-
tive parameter for activation of the CD40 and TNFα
receptors, its concentration was determined upon
stimulation. It was found that levels of IL-6 produced
by HeLaCD40 cells increased significantly when chal-
lenged by both formaldehyde-fixed BHKCD40L and TNFα
but not by formaldehyde-fixed BHKwt. The increase
of IL-6 was up to 2.7 fold with BHKCD40L (p < 0.01) and
5.2 fold with TNFα (p < 0.001) after 16 h incubation with
HeLaCD40 transfected with the HPV18–105 (Fig. 2, b).
This illustrates the efficient stimulation with the func-
tionally active CD40L and TNFα and also confirms
the survival of the transfected HeLaCD40 cells and their
sustained efficiency in releasing IL-6 upon stimulation.
0
10
20
30
40
50
60
70
80
90
100
110
120
130
Medium TNFα BHKWT BHKCD40L
0
10
20
30
40
50
60
70
80
90
100
Medium TNFα BHKWT BHKCD40L
IL
-6
, n
g/
m
l
Re
la
tiv
e
lu
ci
fe
ra
se
-a
ct
ivi
ty
, %
a
b
*
*
*
**
Fig. 2. Effect of CD40L and TNFα on transcription activity
of HPV-LCR: a — transfected HeLaCD40 cells stimulated by TNFα
and formaldehyde-fixed BHK cells for 43 h showed repression
by BHKCD40L and TNFα but not BHKwt (*p < 0.01 vs. BHKwt and
medium) as assessed by the luciferase activity; b — IL-6 was
significantly elevated in the SNs of the corresponding experiment
after 16 h incubation as assessed by ELISA (*p < 0.01 vs. BHKwt
and **p < 0.001 vs. medium)
DISCUSSION
The functional relevance of the CD40/CD40L
interac tions in malignant tumors is variable and likely
context dependent. It has been shown that CD40/CD40L
interac tions or up-regulation of CD40 correlated with
poor prognosis in pancreatic ductal carcinoma [14] and
non-small lung cancer [15], whereas CD40 ligation exerts
antiproliferative effects and apoptosis induction in malig-
nant ovarian tumors [16] and melanoma cell lines [17].
Previous studies also showed that not only TNFα but also
the CD40/CD40L interactions exert a direct cytotoxicity
of HeLaCD40 cells in the presence of a protein inhibitor [9].
In vivo studies revealed that CD40 expression cor-
related with HPV positivity and VEGF expression as well
as microvessel density, which provides explanation for
its high levels in cervical carcinoma and its absence
in cervicitis and normal cervix [18]. Persistent HPV
infection of the cervical epithelium may lead to pro-
gression from cervical dysplasia to cancer over years
or decades. Reso lution of epithelial HPV infection
depends on complex interactions between the HPV-
infected keratinocytes and both host cell-mediated and
humoral responses [19, 20]. Repression of HPV onco-
genes in HeLa cells resulted in the orderly restoration
of dormant tumor suppressor pathways via mobilization
of the p53 and Rb pathways to deliver growth inhibitory
signals to the cells [21]. Interestingly, we could show that
CD40/CD40L interactions and TNFα treatment resulted
Experimental Oncology 38, 22–25, 2016 (March) 25
in repression of the HPV18-p105-promoter in transfected
HeLaCD40.This mechanism could be effective in early
stages of HPV18-infection and could also contribute
to the regression of HPV18-associated high-grade squa-
mous intraepithelial or tumor lesions expressing the HPV
oncogene products. Accordingly, HPV-induced cancer
therapeutic vaccines targeting the E6 and E7 onco-
proteins are combined with adjuvant and costimulatory
anti-CD40 antibodies. The CD40/CD40L interactions and
TNFα treatment exert several antitumor effects, directly
by cytotoxicity and indirectly via repression of oncogenes
and eliciting antigen-specific T cell responses [22].
Various cytokines have been implicated in the
pathogenesis of cervical cancer, among which IL-6 has
received particular attention. Similarly to CD40/CD40L
interactions and TNFα treatment, IL-6 shows a context-
dependent pleiotropic effect as it promotes growth
of certain tumors, such as cervical cancer, whereas
it inhibits the growth of others. Recent studies showed
that IL-6 favors proliferation and metastatic spread and its
high expression in cervical malignant cells corresponds
to bad prognosis. It has also been shown that IL-6 pro-
motes cervical tumorigenesis mainly by activating VEGF-
mediated angiogenesis via a STAT3 pathway [23]. In the
present study, decreased activity of HPV18–105 pro-
moter upon BHKCD40L and TNFα treatment was associated
with the significant increase of IL-6 levels. Although, the
released IL-6 may provide neovascularization, promoting
tumor growth and spread; this may also result in efficient
recruitment of immune cells and better delivery of thera-
peutic cytotoxic drugs. Furthermore, certain cytotoxic
drugs, such as zerumbone, not only induce cell death
and apoptosis but also inhibit IL-6 release [24].
To conclude, the present study further confirms the
antitumor properties of CD40/CD40L interactions and
TNFα treatment in HPV-positive cervical carcinoma.
Boosting of CD40 expression and release of TNFα might
be considered as adjuvant therapies.
ACkNOwLEDGEMENTS
The authors thank Prof. Herbert Pfister, Institute
of Virology of the University of Cologne, for providing
of laboratories and laboratory materials, and Dr. Ger-
trud Steger, Institute of Virology of the University
of Cologne, for the plasmid 4321-luc.
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