smoking and cervical cancer health disparity and cervical cancer health disparity ... curcumin...

Smoking and cervical cancer

health disparity

Subhash C. Chauhan Sanford Research/USD

May 31, 2013

Over-view: • Sanford Heath and Sanford Research/USD

Sanford Health and Sanford Research/USD

• Sanford Health is an integrated health system headquartered in the Dakotas and is now the largest rural, not-for-profit health care system in the nation with locations in 126 communities in seven states

• Sanford Health includes 35 hospitals, 140 clinic locations and nearly 1,200 physicians in 70 specialty areas of medicine.

• With more than 25,000 employees, Sanford Health is the largest employer in North and South Dakota.

• The system is experiencing dynamic growth and development in conjunction with Denny Sanford's nearly $700 million in gifts, the largest ever to a health care organization in America.

• These gifts are making possible the implementation of the several initiatives including global children's clinics, multiple research centers and finding a cure for type 1 diabetes and breast cancer.

http://www.sanfordhealth.org/About

• South Dakota: Population of 824,000

• Native American: 8.9 % (73,300 people)

Cervical Cancer

• Globally: Cervical Cancer is the 2nd most common cancer among women

– Per year, approximately 470,000 new cases, 233,000 deaths

– Majority of cases occur in the developing world

– In many developing countries, cervical cancer is the leading cause of cancer mortality in women

– Screening programs have not been successfully established

• In the United States, during 2011

– ~ 12,710 cases of invasive cervical cancer were diagnosed

– ~ 4,290 women in the US died

• Strong correlation between infection with a high risk genotype of Human Papillomavirus and Cervical Cancer

• Development of the GARDASIL HPV vaccine (targeting HPV 16/18 and 11/6) prevents HPV infection and therefore reduces cervical cancer and genital warts caused by these genotypes of HPV

– Promising, however, cervical cancer screening will still be important…

http://www.cancer.org

Cancer Statistics, 2011. Siegel et al. CA Cancer J Clin 2011;61:212-236

http://www.cancer.org/

Cervical Cancer

1999–2008

Incidence Rates* by Race and Ethnicity, U.S.,

0

5

10

15

20

25

Incidence Mortality

Ra

te p

er 1

00

,00

0

Average Incidence and Mortality of

Cervical Cancer in South Dakota

(2007 - 2009)

Caucasian

American Indian

Mortality Rates* by Race and Ethnicity, U.S.,

US Data retrieved 10/23/12 from http://www.cdc.gov/cancer/cervical/statistics/race.htm

Data from SD Department of

Health (2007 to 2009).

Percentage of U.S. Women Aged 18 Years and

Older Who Have Had a Pap Test in the Last 3

Years by Race and Ethnicity

Available data suggests a similar or higher rate of cervical cancer screening is

obtained for American Indian women living in South Dakota.

Data retrieved 10/23/12 from http://www.cdc.gov/cancer/cervical/statistics/screening.htm

HPV is more commonly detected in AI

women and HPV Positive AI women have

higher rates of abnormal PAP tests

23 %

48 %

0

10

20

30

40

50

60

Caucasian AI

% P

osi

tiv

e

Prevalence of HPV

12%

25%

0

5

10

15

20

25

30

Caucasian AI

% o

f H

PV

Posi

tiv

e W

om

en

HPV Positive Women with an

Abnormal PAP Test

Smoking Rates

12%

49%

0

20

40

60

Caucasian AI

Per

cen

t

Prevalence of Smoking

0%

20%

40%

60%

80%

18-24 25-34 35-44 45-+ 18-24 25-34 35-44 45-+

Caucasian AI

Percent HPV Positive by Age Group

0

5

10

15

20

25

30

6

11

16

18

26

31

33

35

39

40

42

45

51

52

53

54

55

56

58

59

61

62

64

66

67

68

69

70

71

72

73

81

82

83

84

IS39

CP

610

8

% o

f H

PV

in

fect

ion

s

Prevalence of HPV Genotypes (% Based on Positives)

Caucasian AI

Natural Progression of HPV Infection and a Role for Chemoprevention…

Maher et al, Advances in Gynecological Oncology; 2010 Control of Human Papillomavirus gene expression by transcription factors and the upstream regulatory region

• HPV E6/E7 from high risk HPV genotypes are required for the development of cervical cancer

– Most famous pathways

• E6 – degrades p53 (tumor suppressor)

• E7 – interferes with retinoblastoma protein (tumor suppressor)

– Other important pathways

• E6 – activation of telomerase, degradation of proteins with PDZ domains (roles in cell signaling and adhesion)

• E7 - up-regulation of AKT pathway, and interactions with various cell signaling molecules (cyclin A and E, p27, p21 etc.)

• Reducing the amount of HPV E6/E7 should be beneficial in

interrupting the development of invasive cervical cancer.

Curcumin (diferuloyl

methane)

Maheshwari, et al. 2006. Life Sciences.

0

20

40

60

80

100

120

10 20 40 80

% C

han

ge

Curcumin (µM)

SiHa

SW756

HeLa

C33A

Caski

*

*

*

*

*

A. B.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 5 10 15

Clo

nogen

ic P

ote

nti

al

Curcumin (µM)

Caski

SiHa

*

*

*

*

C.

DMSO Control 20 µM Curcumin 40 µM Curcumin

Collagen Collagen Collagen

D.

DMSO 2 µM Curcumin

5 µM Curcumin 10 µM Curcumin

Curcumin treatment suppresses cervical cancer cell

growth in monolayer and organotypic raft culture systems

Maher et al, Molecular Carcinogenesis, 50:47–57 (2011)

0

20

40

60

80

100

120

DMSO Curcumin PLGA Nanocurcumin

% C

han

ge 5

10

15

A PLGA nano-formulation of Curcumin effectively

suppresses cervical cancer cell growth

Micromolar concentrations

MTS assay 24 hours after treatment

A. B. C. DMSO 10 µM

20 µM 40 µM

PARP

Caspase 3

Caspase 9

D 10 20 40

β-actin

35 kDa

17/19 kDa

89 kDa 116 kDa

47 kDa

37 kDa

17 kDa

45 kDa

0

10

20

30

40

50

DMSO 10 20 40

% D

ead

Cel

ls

µM Curcumin

*

*

24 hr 24 hr

7AAD Staining 24 hr

*p<0.05

D = DMSO Maher et al, Molecular Carcinogenesis, 50:47–57 (2011)

Curcumin treatment induces apoptosis in cervical

cancer cells via caspase-mediated signaling.

A.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

DMSO 10 20 40 DMSO 10 20 40

Exp

ress

ion

Lev

el

* * * *

24 hr 48 hr

D 10 20 40 D 10 20 40

24 hr 48 hr

β-actin

HPV E7

45 kDa

15 kDa

Protein

*p<0.05

0.0

0.2

0.4

0.6

0.8

1.0

1.2

DMSO 10 20 40

Exp

ress

ion

Lev

el

HPV E6 mRNA

* *

*

B.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

DMSO 10 20 40

Exp

ress

ion

Lev

el

Curcumin (µM)

HPV E7 mRNA

* *

*

RNA: 6 hr after addition of curcumin

*p<0.005

D = DMSO Maher et al, Molecular Carcinogenesis, 50:47–57 (2011)

Curcumin treatment represses the expression of

HPV oncogenes E6 and E7

MCS

HPV 16 URR

HPV 16 URR Luciferace Reporter

0

0.2

0.4

0.6

0.8

1

1.2

DMSO CurcuminN

orm

ali

zed

Rep

ort

er A

ctiv

ity

Curcumin Treatment

Curcumin treatment represses the activity of the

HPV16 URR (upstream regulatory region)

HPV URR activity

in cervical cancer cells (25 µM Curcumin for 6 hr) Plasmid Transfection

(48 hr)

Normalize data to control plasmid

(Tk-CLuc) and DMSO

Collect supernatant and analyze

for Luciferace activity

0

5

10

15

20

D 10 20 40

RE

L

p53

0

2

4

6

D 10 20 40

RE

L

pRb

0

5

10

15

D 10 20 40

RE

L

PTPN13

D 10 20 40 24 hr

p53

Rb

β-actin

PTPN13

45 kDa

277 kDa

110 kDa

53 kDa

Curcumin restores the expression of

tumor suppressor proteins: p53, Rb, PTPN13 (each known to be degraded when HPV E6 or E7 is expressed)

D = DMSO

REL: relative expression level Maher et al, Molecular Carcinogenesis, 50:47–57 (2011)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

DMSO 5 10

Mo

tili

ty I

nd

ex

Curcumin (µM)

*

*

T=0hr

T=6hr

DMSO 5 µM Curcumin 10 µM Curcumin

Curcumin treatment inhibits the motility of

cervical cancer cells

p<0.005


Smoke Carcinogen: Benzo[a]pyrene (BaP)

• Cigarette smoking is known to be a risk factor for cervical cancer

– However, the molecular link between smoking and HPV is unknown

• BaP is a polycyclic hydrocarbon. These compounds are generated by burning carbon containing materials, such as:

– Tobacco, Charbroiled Meat, Fried Food (especially when the same oil is used repeatedly, Wood

• BaP is detected in cervical mucus of women who smoke • Melikian AA, Sun P, Prokopczyk B, et al. Identification of benzo[a]pyrene metabolites in cervical mucus and DNA

adducts in cervical tissues in humans by gas chromatography-mass spectrometry. Cancer Lett 1999; 146: 127-34.

• McCann MF, Irwin DE, Walton LA, Hulka BS, Morton JL, Axelrad CM. Nicotine and cotinine in the cervical mucus of

smokers, passive smokers, and nonsmokers. Cancer Epidemiol Biomarkers Prev 1992; 1: 125-9.

12%

49%

0

20

40

60

Caucasian AI

Per

cen

t

Prevalence of Smoking

Tobacco smoke compound BaP up-regulates the expression of HPV

oncogenes, NFκB and AP1 but is suppressed by curcumin treatment

0.0

0.5

1.0

1.5

2.0

2.5

DMSO 0.1 1.0 DMSO 0.1 1.0

*

*

* * *

BaP + Curcumin (20 µM)

BaP (µM)

BaP

A. B.

HPV E7

β-actin

0 0.1 1.0 BaP (µM) 0 0.1 1.0 Curcumin - - - + + +

45 kDa

15 kDa

RE

L

NFκB and AP1

0

0.5

1

1.5

2

NFkB: p50 AP1: c-Fos

RE

L

DMSO

10 nM BaP

100 nM BaP

0

0.2

0.4

0.6

0.8

1

1.2

100 nM BaP 100 nM BaP + 20

µM Cur

RE

L

C.

*

NFkB: p50


Schematic model of increased oncogenic signals via HPV E6/E7 oncoproteins and molecular

effect of curcumin on HPV associated cellular events.

Top: HPV oncoproteins degrade tumor suppressor proteins, increasing the risk of

developing cancer. BaP increases the expression of HPV oncoproteins, potentially

increasing oncogenic signals and even in the presence of only a few copies of HPV

genome.

Bottom: Curcumin specifically inhibits the expression of HPV oncoproteins, even in the

presence of BaP, thereby reducing the oncogenic signals and potentially inhibiting

cancer development.


BaP exposure activates the AhR pathway

increasing CYP1A1 expression

AhR

DM

SO

0.1

µM

Ba

P

No

Pri

ma

ry

Co

ntr

ol

Composite

Caski

6 hour

0

10,000

20,000

30,000

40,000

50,000

60,000

70,000

80,000

90,000

100,000

DMSO BaP 100 nM BaP 500 nM

Mea

n F

l. I

nte

nsi

ty

500 nM BaP

100 nM BaP

DMSO

No Primary Control

Representative data from 3 independent experiments

AhR is aberrantly expressed and localized in pre-

neoplastic and invasive cervical cancer tissues

0

5

10

15

Normal CIN I CIN II CIN III Invasive

MC

S

AhR Expression: Cytoplasmic

0

2

4

6

8

Normal CIN I CIN II CIN III Invasive

MC

S

AhR Expression: Nuclear

normal (n=10), cervical intraepithelial neoplasia I (CIN I, n=10), CIN II (n=10), CIN III (n=6), and invasive cervical cancer (n=15).

200x

A) Stable tdTomato expression in cervical cancer cells

Red Fluorescence

Phase Contrast

WT 10 x 106 cells 2.5 x 106 cells 1.25 x 106 cells

B) In vivo detection of tdTomato expressing cervical cancer cells

Development of an orthotopic cervical cancer mouse model

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

week 1 week 2 week 3 week 4

Mea

n F

luo

resc

en

t In

ten

sity

Week 1 Week 2 Week 3 Week 4 Control

Mouse

Growth of orthotopic cervical tumors

Mouse injected with

2.5x106TdTomato expressing cells

Cervical Cancer Orthotopic Mouse Model

PBST PLGA-curcumin Curcumin PLGA

Treatment of orthotopic cervical tumors

Mice were treated by intratumoral injection 2x’s per week

• BaP increases oncogenic signals from HPV infection

• Curcumin may inhibit this signaling in vivo

• Smoke exposure may modulate the local immune response, favoring

persistent HPV infection and development of cervical cancer

Curcumin

Loss of p53, Rb,

PTPN13 and

other changes

XAP2

BaP

AhR protein complex

AhR

ARNT

Smoke Exposure Containing BaP

E6 and E7

expression

CYP1A1

and

others

Increased

oncogenic

signaling

Hsp 90 Hsp 90

Increase

mediated

by BaP

HPV

Curcumin

XAP2

Acknowledgements Members of the Chauhan Lab

– Dr. Diane Maher

– Dr. Mohammed Zaman

– Neeraj Chauhan

– Mara Ebeling

– Dr. Brij Gupta

– Dr. Mohan Yallapu

– Dr. Sheema Khan

– Dr. Rishi Gara

– Dr. Sikender Mohammed

– Emily Gaster

– Amanda Schaefer

– Emmylu O’Donnell

• Dr. Maria Bell

• Cancer Biology Research Center

• Sanford Research/USD

Funding sources:

• NIH 1U01CA162106-01A1

• NIH COBRE P20RR024219

smoking and cervical cancer health disparity and cervical cancer health disparity ... curcumin...

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