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A new topical treatment for HPV-induced neoplasia. Gary Disbrow Astrid Baege Kate Kierpiec Hang Yuan Dan Hartmann Richard Schlegel. Georgetown University. HPV-induced neoplasia.

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A new topical treatment for HPV-induced neoplasia

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A new topical treatment for hpv induced neoplasia

A new topical treatment for

HPV-induced neoplasia

Gary Disbrow

Astrid Baege

Kate Kierpiec

Hang Yuan

Dan Hartmann

Richard Schlegel

Georgetown

University


A new topical treatment for hpv induced neoplasia

HPV-induced neoplasia

  • 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.


Iron as a target

Iron as a target

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

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


A new topical treatment for hpv induced neoplasia

Dihydroartemisinin

Artemisia annua

The structure of DHA


A new topical treatment for hpv induced neoplasia

Endoperoxide bridge

Fe++

OH

OH

OH-

OH

Iron-dependent activity of DHA

Dihydroartemisinin

DNA damage


A new topical treatment for hpv induced neoplasia

Cell morphology after treatment with DHA

HCX control

HCX 3d 25 µM DHA

HeLa control

HeLa 3d 25 µM DHA


A new topical treatment for hpv induced neoplasia

HeLa cells treated with artemisinin and

derivatives

120

100

80

Artemisinin

% Cell Survival

60

Artesunate

DHA

40

20

0

0

10

20

30

40

50

60

m

M


A new topical treatment for hpv induced neoplasia

Normal cervical cells treated with

artemisinin and derivatives

120

100

80

Artemisinin

% Cell Survival

60

Artesunate

DHA

40

20

0

0

10

20

30

40

50

60

m

M


A new topical treatment for hpv induced neoplasia

Cell lines treated with DHA

120

100

HCX

80

HCX-E6E7 p5

HCX-E6E7 p50

% Cell Survival

60

SiHa

40

Caski

HeLa

20

0

0

10

20

30

40

50

60

m

M DHA


A new topical treatment for hpv induced neoplasia

Cell lines treated with artesunate

120

HCX

100

HCX-E6E7 p4

80

HCX-E6E7 p45

% Cell Survival

60

SiHa

Caski

40

HeLa

20

0

0

10

20

30

40

50

60

m

M Artesunate


A new topical treatment for hpv induced neoplasia

Cell killing at higher concentrations

of artemether


A new topical treatment for hpv induced neoplasia

Chelation of iron inhibits killing of

HeLa cells by DHA

120

100

m

0

M DFOM

80

m

25

M DFOM

% Cell Survival

60

m

100

M DFOM

m

200

M DFOM

40

20

0

0

25

50

75

100

125

150

175

m

M DHA


A new topical treatment for hpv induced neoplasia

Measuring reactive oxygen species

with DCF

488 nm

Cellular

esterases

H2O2, OH-

Non-fluorescent,

reduced form

Fluorescent,

oxidized form

Cell membrane

570 nm


A new topical treatment for hpv induced neoplasia

DHA

DFOM + DHA

Cell Count

FITC-A

Induction of reactive oxygen species

in HeLa cells

Untreated

0 mM DHA

Pretreated with 150 mM DFOM

5 mMC-DCF-DA

25 mM DHA

50 mM DHA


A new topical treatment for hpv induced neoplasia

Primary cervical cells

0 uM DHA

10 uM DHA

25 uM DHA

50 uM DHA

HeLa cells

DHA induces apoptosis in HeLa cells but not in primary cervical cells


A new topical treatment for hpv induced neoplasia

Cleaved Caspase 3

100 +

150 DFOM

100 +

150 DFOM

0

100

0

50

100

19 kD

47 kD

b-Actin

100 +

150 DFOM

100 +

150 DFOM

0

50

100

0

50

100

DHA activates caspases in the mitochondrial pathway and induces PARP cleavage in HeLa cells

Cleaved Caspase 9

36 kD

47 kD

b-Actin

Cleaved Caspase 7

Cleaved PARP

19 kD

81 kD

47 kD

47 kD

b-Actin

b-Actin


Canine oral papillomavirus copv as a model for human disease

Canine oral papillomavirus (COPV)as 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).


A new topical treatment for hpv induced neoplasia

Dogs Challenged

with COPV-1

All tumors

had regressed

5 wks

3 wks

Start treatment

with DHA or DMSO

24 hrs later

Tumor formation

started

Stop

treatment

Canine oral papillomavirus model


A new topical treatment for hpv induced neoplasia

Dogs Challenged

with COPV-1

Tumor formation

started

All tumors

had regressed

3 wks

5 wks

Start treatment

with DHA or DMSO

24 hrs later

Stop

treatment

Viral challenge + DHA

Viral challenge + DMSO

In vivo activity of DHA

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.


A new topical treatment for hpv induced neoplasia

Tumor formation in dogs

*Tumors regressed two weeks earlier

than the DMSO-treated animals


A new topical treatment for hpv induced neoplasia

Normal

Dog Sera

1

2

3

4

5

6

DMSO

DHA

DHA does not prevent early papillomavirus infection

Anti-L1 capsid protein antibody titers

0.6

0.5

0.4

OD 450

0.3

0.2

0.1

0


A new topical treatment for hpv induced neoplasia

DHA does not inhibit early viral protein

expression in vitro

HCX

HeLa

DHA mM

0

50

100

100

0

50

100

100

DFOM mM

150

150

p53

b-actin

E7

b-actin


A new topical treatment for hpv induced neoplasia

Summary

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 the

topical treatment of mucosal HPV infections

1. Artemisinin derivatives are currently approved in humans

for the systemic treatment of malaria

2. DHA is hydrophobic and readily penetrates mucosal

surfaces

3. In addition to inducing apoptosis, artemisinin derivatives

have anti-angiogenic activity


A new topical treatment for hpv induced neoplasia

The first-generation papillomavirus vaccine:

a virus-like particle (VLP)

Virions

VLP


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