A new topical treatment for A new topical treatment for HPV-induced neoplasiaHPV-induced neoplasia
GeorgetownUniversity
Gary DisbrowAstrid BaegeKate KierpiecHang YuanDan HartmannRichard Schlegel
•High-risk HPVs are the etiologic agents in 99% of cervical cancers (Walboomers 1999) and also have a role in a subset of oral, anal, esophageal, and epidermal carcinomas.
•Low-risk HPVs induce benign tumors at many anatomic sites, including those of mucosal and epidermal origins.
•To study mucosal papillomavirus infections and to evaluate potential therapies (including vaccines), we have utilized the canine oral papillomavirus model.
HPV-induced neoplasiaHPV-induced neoplasia
Iron as a targetIron as a target
Artemisinin is the active principle of the Chinese herb, Artemisia annua, and is currently used clinically for treating drug-resistant malaria.• toxicity is dependant upon interactions with iron • DHA is a metabolic intermediate of artemisinin
Many HPV-expressing cells, including cervical cancer cells, overexpress the transferrin receptor
•Potential for higher levels of intracellular iron•Distinction between cancer and normal cells
DihydroartemisininArtemisia annua
The structure of DHAThe structure of DHA
Iron-dependent activity of DHAIron-dependent activity of DHA
Endoperoxide bridge
OH
OH
OH
OH-
Fe++
DNA damage
Dihydroartemisinin
HCX control HCX 3d 25 µM DHA
HeLa control HeLa 3d 25 µM DHA
Cell morphology after treatment with DHACell morphology after treatment with DHA
HeLa cells treated with artemisinin and HeLa cells treated with artemisinin and derivativesderivatives
0 10 20 30 40 50 600
20
40
60
80
100
120
ArtemisininArtesunateDHA
M
% C
ell S
urv
iva
l
Normal cervical cells treated with Normal cervical cells treated with artemisinin and derivativesartemisinin and derivatives
0 10 20 30 40 50 600
20
40
60
80
100
120
ArtemisininArtesunateDHA
M
% C
ell S
urv
ival
Cell lines treated with DHACell lines treated with DHA
0 10 20 30 40 50 600
20
40
60
80
100
120
HCXHCX-E6E7 p5HCX-E6E7 p50SiHa
Caski
HeLa
M DHA
% C
ell S
urv
ival
Cell lines treated with artesunateCell lines treated with artesunate
0 10 20 30 40 50 600
20
40
60
80
100
120
HCXHCX-E6E7 p4HCX-E6E7 p45SiHaCaskiHeLa
M Artesunate
% C
ell
Su
rviv
al
0
10
20
30
40
50
60
70
80
90
100
0 50 100 150 200 250 300 350 400
Artemether [μM]
% c
ell
su
rviv
al HCX
C33A
SiHa
Caski
Hela
Cell killing at higher concentrations Cell killing at higher concentrations of artemetherof artemether
Chelation of iron inhibits killing of Chelation of iron inhibits killing of HeLa cells by DHAHeLa cells by DHA
0 25 50 75 100 125 150 1750
20
40
60
80
100
120
0 M DFOM25 M DFOM
100M DFOM
200 M DFOM
M DHA
% C
ell S
urv
ival
Cel
lula
r e
ster
ases
Non-fluorescent,reduced form
Fluorescent,oxidized form
H2O
2, O
H-
Cell membrane
488 nm
570 nm
Measuring reactive oxygen speciesMeasuring reactive oxygen species with DCFwith DCF
FITC-A
Cel
l Cou
ntDHA DFOM + DHA
Induction of reactive oxygen species Induction of reactive oxygen species in HeLa cellsin HeLa cells
Untreated
0 M DHA
25 M DHA
50 M DHA
Pretreated with 150 M DFOM5M C-DCF-DA
DHA induces apoptosis in HeLa cells but not DHA induces apoptosis in HeLa cells but not in primary cervical cellsin primary cervical cells
Primary cervical cells
HeLa cells
10 uM DHA0 uM DHA 25 uM DHA 50 uM DHA
DHA activates caspases in the mitochondrial pathway DHA activates caspases in the mitochondrial pathway and induces PARP cleavage in HeLa cellsand induces PARP cleavage in HeLa cells
-Actin
Cleaved Caspase 3
19 kD
47 kD
-Actin
Cleaved Caspase 9
36 kD
47 kD
100 +
150 DFOM100500
81 kD
-Actin
Cleaved PARP
47 kD
19 kD
-Actin
47 kD
Cleaved Caspase 7
0 50 100100 +
150 DFOM100500100 +
150 DFOM
100 0100 +
150 DFOM
Canine oral papillomavirus (COPV)Canine oral papillomavirus (COPV)as a model for human diseaseas a model for human disease
• Canine oral papillomavirus infects and induces tumors at mucosal sites, mimicking the biology of mucosal papillomavirus infections.
• As in human disease, tumor induction and growth is markedly affected by host immune status.
• In animals with persistent infection, carcinomas develop after 2 years and metastasize widely.
• The canine model has been used to provide “pre-clinical” data prior to phase trials of human vaccines (MedImmune and GSK).
Dogs Challenged with COPV-1
Start treatmentwith DHA or DMSO
24 hrs later
3 wks
Tumor formationstarted
All tumorshad regressed
5 wks
Stoptreatment
Canine oral papillomavirus modelCanine oral papillomavirus model
Dogs Challenged with COPV-1
Start treatmentwith DHA or DMSO
24 hrs later
3 wks
Tumor formationstarted
All tumorshad regressed
5 wks
Stoptreatment
Viral challenge + DHAViral challenge + DMSO
Application method
Dogs were treated daily with 100 ul of DMSO or DHA. The DHA was at a concentration of 78.13 mM (stock). Every third day, the dogs were placed
under light anesthesia to ensure a more thorough treatment.
In vivo activity of DHAIn vivo activity of DHA
Dog IDDMSO or
DHARight
Side
Left
Side
1 DMSO + +
2 DMSO + +
3 DMSO + +
4 DHA - -
5* DHA + +
6 DHA - -
Tumor formation in dogsTumor formation in dogs
*Tumors regressed two weeks earlierthan the DMSO-treated animals
NormalDog Sera
1 5 63 42
DMSO DHA
OD
450
Anti-L1 capsid protein antibody titers
DHA does not prevent early papillomavirus infectionDHA does not prevent early papillomavirus infection
0
0.1
0.2
0.3
0.4
0.5
0.6
0 0100 100 100 10050 50
150 150
DHA M
DFOM M
HCX HeLa
p53
-actin
E7
-actin
DHA does not inhibit early viral protein DHA does not inhibit early viral protein expression in vitroexpression in vitro
SummarySummary
DHA induces rapid, iron-dependent, p53-independent apoptosis in PV-expressing epithelial cells in vitro
DHA prevents the formation of PV-induced tumors in vivo
DHA has several features which make it suitable for thetopical treatment of mucosal HPV infections
1. Artemisinin derivatives are currently approved in humansfor the systemic treatment of malaria
2. DHA is hydrophobic and readily penetrates mucosalsurfaces
3. In addition to inducing apoptosis, artemisinin derivatives have anti-angiogenic activity
Virions
The first-generation papillomavirus vaccine:The first-generation papillomavirus vaccine:a virus-like particle (VLP)a virus-like particle (VLP)
VLP