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