Physalin A inhibit inflammatory effects through the MAPK pathway

(1)

ANTI-INFLAMMATORY ACTIVITIES OF PHYSALIN A

FROM

PHYSALIS ANGULATA

THROUGH THE

INHIBITION OF MMP-9, NF-

ΚB, AND MAPK

ACTIVATION

IN VITRO

AND

IN VIVO

WEN-TSONG, HSIEH

文

聰

謝

Department of Pharmacology,

School of Medicine,

China Medical University

Taichung, Taiwan

2012.11.15

GSU/CMU Biotech symposium 2012

First part

Anti-cancer research

in Our Lab

Background of our lab. in anti-cancer research

_{Risk factors of Oral Cancer}

• Betel nut

• Tobacco

• Alcohol

• Diet and nutrition,

• Ultraviolet light,

• HPV infection

(the Institute of Medicine, 2007)

(J Oral Pathol Med, 1989)

(Global cancer statistics, 2002)

(Ann Oncol, 2003)

4

Michael Ericksen

Dean, institute of public Health

5 Anticancer research in TCM and folk herbs

1. 山芙蓉 Hibiscus taiwanensis

2.山豆根 Sophora subprostrata

3. 龍葵 Solanum nigrum

4. 香茅 Cymbopogon citratus

5. 艾草 Artemisia indica

6. 一枝黃花 Solidago decurrens

7. 鉤藤 Viburnum odoratissimum

8. 苦蘵 Physalis angulata

9. 金剛纂 Euphorbia antiquorum

10………

2001 Lee et al., Effects and mechanisms of PAon cell death in human lung squamous cell carcinoma. 2001 Lee et al., Effects and mechanisms of PAon cell death in human lung squamous cell carcinoma. 1992 Chiang et al., Inhibitory effects of physalin B and physalin F on various

human leukemia cells

in vitro. 1992 Chiang et al., Inhibitory effects of physalin B and physalin F on various

human leukemia cells

in vitro. 2002 Kuo et al., The antiproliferative activity of PAis through p53-dependent and p21-dependent apoptotic pathway in human hepatoma cell lines.

2002 Kuo et al., The antiproliferative activity of PAis through p53-dependent and p21-dependent apoptotic pathway in human hepatoma cell lines.

2001 Ismail et al., A novel cytotoxic flavonoid glycoside from Physalis angulata. 2001 Ismail et al., A novel cytotoxic flavonoid glycoside from Physalis angulata. 2004 Wu et al., Antihepatoma activity of PA

and P. peruviana extracts and their

effects on apoptosis in human Hep G2 cells.

2004

Wu et al.,

Antihepatoma activity of PA

and P. peruviana extracts and their

effects on apoptosis in human Hep G2 cells. 2003 Makino et al., Cytotoxic activity of physalinspossessing modified skeletal structures against HeLa cells. 2003 Makino et al., Cytotoxic activity of physalinspossessing modified skeletal structures against HeLa cells. 2001s 1992s 2005 Vieira AT et al., Mechanisms of the anti-inflammatoryeffects of the natural secosteroids physalins in a model of intestinal ischaemia and reperfusion injury.

2005

Vieira AT et al., Mechanisms of the anti-inflammatoryeffects of the natural secosteroids physalins in a model of intestinal ischaemia and reperfusion injury.

2006

Magalhs HI, et al.,

In-vitroand in-vivo antitumour activityof physalins B and D from Physalis angulata.

2006

Magalhs HI, et al.,

In-vitroand in-vivo antitumour activityof physalins B and D from Physalis angulata.

2007 Castro DP et al., Immune depression in Rhodnius prolixus by seco-steroids, physalins. 2007 Castro DP et al., Immune depression in Rhodnius prolixus by seco-steroids, physalins. 2002s 2003s 2004s 2005s 2006-2011 2012s2007s

Physalis angulata

(Solanaceae)

Usage: Herb tea

涼茶

(清涼退火)

Extinguish the fire inside the body

(2)

7

8 Isolation physalins from

physalis angulata

_Methods

Morphology

MTT assay

Cell cycle analysis (Flow

cytometry) Western blot

1. Proliferation

3. Metastasis

Migration assay

-

Wound healing assay

-

Transwell migration assay

Matrigel invasion assay

Confoal image

Western blot

2. Apoptosis

Flow cytometry

-

Ca

++

_production

-

Mitochondrial membrane potential (∆Ψ

DAPI stain

DNA ladder assay

Western blot

Physalin A on cell cycle analysis by flow cytometer

Physalin A induced G2/M arrest and apoptosis in HSC-3 cells in

dose & time dependent.

Result-G2/M arrest & apoptosis

3.13 µM 6.25 µM 12.5 µM 17 control 0.78 µM 1.56 µM HSC-3 cell Physalin A [uM] Control0.39 0.78 1.56 3.13 6.25 12.5 C e ll n u m b er ( % ) 0 10 20 30 40 50 60 70 80 G0/G1 G2/M S Apoptosis cdc2 60kDa 0 0.39 0.78 1.56 3.13 6.25 12.5 (µM) 58kDa 53kDa 27kDa 34kDa cyclinB1 cyclinA p53 p27 42kDa Actin Wee1 105kDa

HSC-3

PA 1.0 0.5 0.3 0.4 0.5 0.5 0.7 1.0 0.9 0.8 0.7 0.6 0.6 0.2 1.0 1.2 1.1 1.2 1.1 0.9 0.9 1.0 0.6 0.4 0.6 0.7 1.0 1.1 1.0 1.5 1.7 1.9 1.8 2.0 2.0 1.0 0.5 0.3 0.3 0.2 0.3 0.1

Physalin A

induced G2/M arrest

with

decreasing cyclin A, cyclin B1

and

cdc2

activity, but

increasing wee1, p27

and

p53

levels.

G2/M phase arrest

cyclinA/B1

P

p53

PA

Wee1

p27

Physalin A on G2/M phase of cell cycle relative proteins

19

Result-G2/M arrest

(3)

13 Effects of PA on mRNA expression of cyclin

A and cyclin B1 in HSC-3 cells.

Physalin A

induced G2/M arrest

with

decreasing mRNA expression in

cyclin A

levels, dose dependently .

Methods

Morphology

MTT assay

Cell cycle analysis (Flow

cytometry) Western blot

1. Proliferation

3. Metastasis

Migration assay

-

Wound healing assay

-

Transwell migration assay

Matrigel invasion assay

Confoal image

Western blot

2. Apoptosis

Flow cytometry

-

Ca

++

_production

-

Mitochondrial membrane potential (∆Ψ

DAPI stain

DNA ladder assay

Western blot

(µM)

500 bp

400 bp

200 bp

100 bp

300 bp

To analyze the DNA

fragmentation, HSC-3 cells were

treated with Physalin A for 24 hr.

Fragmented DNA was extrcted

and analyzed on 2 % agarose gel

electrophoresis with containing

EtBr.

Physalin A could induced

DNA

damage with DNA ladder assay

(apoptosis) in HSC-3 cells.

Physalin A on DNA damage with DNA ladder assay

Result-DNA fragmentation

2 6

Cells were harvested after 24 h of PA treatment. After fixing, the

cells were stained with DAPI. Stained nuclei were then observed

under a fluorescent mircroscope.

PA induced DNA condensation in

HSC-3 cells.

Effects of PA on DNA condensation with DAPI stain

3.13 µM

6.25 µM

12.5 µM

control

0.39 µM

0.78 µM

1.56 µM

200 x 2 4

(Negative control)

0.39 µM

0.78 µM

1.56 µM

DMSO

(positive control)

5 µM H

2

O

2

Physalin A on DNA damage by Comet assay

HSC-3 cells were treated

with Physalin A (0.39, 0.78,

1.56 µg/ml) for 24 hr and

applied to

Single cell

electrophoresis

Physalin A induced DNA

damage dose dependent by

the Comet assay in HSC-3

cells

Result-DNA damage Phsalin A [µg/ml] T a il momen t (%) 2 8

Effects of PA-42 on Ca

2+

_{production by flow cytometry}

Result-Ca 2+production

24 hr

0 hr

1 hr

6 hr

12 hr

6 hr

0 hr 1 hr 6 hr 12 hr 18 hr 24 hr Time (hr) Ca 2+ p roduction (%) 21

Cells were harvested

various hour of PA

PA-42 could increase

Ca

++

_{production at 6-12}

(4)

Measurement of mitochondrial membrane potential (∆Ψ

m

)

Ca

2+

_{was determined by Indo 1/AM flow cytometry.}

Mitochondrial energization was determined by the retention of the dye

DiOC

6

3'-dihexyloxacarbocyanine iodide

flow cytometry.

2 0

Method-Ca2+and MMPanalysis

Measurement of Ca

2+

(Cell Death Differ, 2007)

pro-casp-3 17kDa 32kDa Bcl-2 26kDa 0 0.39 0.78 1.56 3.13 6.25 12.5 (µM) AIF 57kDa Actin 42kDa 21kDa Bax Bid Fas 40kDa 22kDa PA cleaved casp-3 1.0 2.2 2.9 5.3 6.8 7.1 7.1 1.0 1.5 1.8 2.1 2.2 2.1 2.1 1.0 1.2 1.3 1.4 1.2 1.1 0.9 1.0 0.9 0.7 0.8 0.8 0.8 0.8 1.0 1.0 1.0 1.1 1.0 1.0 1.0 1.0 1.1 1.1 1.3 1.3 1.5 1.2 1.0 1.1 1.1 1.4 1.7 1.9 1.9 HSC-3

Physalin A

triggered apoptosis

with an

increase in Fas, Bax, Bid,

and cleaved caspase-3

but

a decrease in Bcl-2.

Fas

Ca

2+

Bax Bid

Apoptosis

Bcl-2

pro-caspase-3

cleaved caspase-3

PA

_MMP

(∆Ψm)

AIF

Ca

2+

Effects of Physalin A on apoptotic proteins

AIF: apoptosis-inducing factor MMP : mitochondrial membrane

i l

2 9

Result- Apoptosis proteins

21

Channels (FL2-A) 0 50 100 150 200 250 0 50 100 150 200 Channels (FL‐2) Num b er 100 200 300 400 PA 6.0 μM Pifithrin-α 20 μM Channels (FL2-A) 0 40 80 120 160 200 0 50 100 150 200 Channels (FL‐2) Num b er 100 200 300 400 PA 6.0 μM Pifithrin-α 40 μM Channels (FL2-A) 0 50 100 150 200 250 0 50 100 150 200 Channels (FL‐2) Num b er 100 200 300 400 PA 6.0 μM Pifithrin-α 10 μM Channels (FL2-A) 0 40 80 120 160 200 0 50 100 150 200 Channels (FL‐2) Num b er 100 200 300 400 PA 6.0 μM Pifithrin-α 80 μM PA 6.0 μM Pifithrin-α 0 μM Num b er 100 200 300 400 0 50 100 150 200 250 0 50 100 150 200 Channels (FL‐2) PA 0 μM Pifithrin-α 0 μM Channels (FL2-A) 0 40 80 120 160 200 0 50 100 150 200 Channels (FL‐2) Num b er 100 200 300 400 PA 6.0 μM - + + + + + - - 10 20 40 80 PTF-α (μΜ ) C ell num ber (% ) 0 10 20 30 40 50 60 70 G0/G1 phase G2/Mphase S phase Apoptosis a a b b a b a a b b

PA on 2D-gel

Protein Name Accession Number pI MW _(Da)

264 Heat shock 70kDa protein 8 [Homo

sapiens]

gi|16741727 5.37 70854

268 HSP70-2 [Homo sapiens] gi|4529892 5.48 69982

RESULT –HSP

264

268 HSP90

HSP70-2

β-actin

90 kDa

40 kDa

42 kDa

2.58

1.97

1.80

1.04

1.00

1.11

1.13

0.95

1.00 PA(μM)

0 1.6 3.1 6.3 12.5

Methods

Morphology

MTT assay

Cell cycle analysis (Flow

cytometry) Western blot

1. Proliferation

3. Metastasis

Migration assay

-

Wound healing assay

-

Transwell migration assay

Matrigel invasion assay

Confoal image

Western blot

2. Apoptosis

Flow cytometry

-

Ca

++

_production

-

Mitochondrial membrane potential (∆Ψ

DAPI stain

DNA ladder assay

Western blot

PA in 2D-gel analysis

Protein Name Accession _Number pI MW (Da)

352 alpha-tubulin [Homo sapiens] gi|340021 4.94 50120

369 Tubulin, beta [Homo sapiens] gi|14043231 4.78 49639

480 Nucleophosmin (nucleolar

phosphoprotein B23, numatrin) [Homo sapiens]

gi|16876872 4.64 32555

352

480

369

(5)

Effects of PA-42 on

cell migration

by wound healing assay

PA-42

inhibited wound healing

significant

&

without cytotoxicity.

Result-wound healing migration

Serum-free medium for 24 hr Seeding cells wound Migration PA-42 33 Time (hr) Wo und range (µm )

Result-transwell migration assay

0.39 µg/ml

0.78 µg/ml

1.56 µg/ml

control

Upper chamber (1%FBSmedium) Lower chamber (10% FBS medium) Basement membrane layer

Crystal violet Staining

Physalin A for 24 hr inhibited ~50% migration

without significant cytotoxicity HSC-3 cells

PA Concentration (µM) In h ib ition rate (% of co n tro l) 3 4

Physalin A on cell migration by Transwell Migration assay

Effects of PA 0.75~1.56 µM on matrigel invasion assay

Result-matrigel invasion assay

Upper chamber (1%FBS medium)

Lower chamber (10%FBSmedium) Coated 50 µl matrigel

Crystal violet Staining

HSC-3 cells at

IC

10

dose of PA for 24 hr

inhibited ~35% invasion

without

significant cytotoxicity by the ImageJ

freeware analyzed.

PA

control

0.39 µM

0.78 µM

1.56 µM

35 Cell count mean (± SD) Inhibition rate (% of control) Control 782.33 (± 34.27) 0% 0.75 617.67 (± 55.99) 21.05% 1.5 536.33 (± 17.93) 31.44% 3 497.67 (± 4.04) 36.39% 0 0.39 0.78 1.56 PA [μM] In vasio n cell s (% ) 0 200 400 600 800 0 0.39 μM 0.78 μM 1.56 μM * ** ***

Red : ®-Actin (rhodamine) Blue : DAPI (DNA stain) control

merge merge

Physalin A 1.56 µg/ml

Physalin

reversed cytoskeleton

from

mesenchymal-like

to

epithelial-like

with

increased E-cadherin (epithelial marker)

and

decreased α-smooth muscle

actin (mesenchymal marker ) in HSC-3 cells.

Green : E-cadherin (FITC) Blue : DAPI (DNA stain) control

merge merge

Physalin A 1.56 μg/ml

Green : α-smooth muscle actin (FITC) Blue : DAPI(DNA stain) control

merge merge

Physalin A 1.56 μg/ml

Physalin A on metastasis relative proteins by confocal image

culture slides Cells PA cancer cell 2 2 confocol microscope (630x) 3 6 Result-cytoskeleton proteins 0 0.39 0.78 1.56 (µM) MMP-2 55kDa β-actin 42kDa GRB2 24kDa HSC-3 PA 1.0 0.7 0.8 0.7 1.0 0.9 0.8 0.7

Physalin A

inhibited metastasis

with a decrease in MMP-3

and GRB2.

Physalin A on metastasis relative proteins

MMP-3 : matrix metalloproteinase-3 GRB2 : growth factor receptor-bound protein 37 Result-metastasis proteins

Epithelial-like

MMP-2

migration

invasion

Mesenchymal-like

E-cadherin

GRB2

α-smooth muscle Actin

metastasis

30

Gross and histopathological findings of hamster’s pouch in (7,12-dimethylbenz

[α]anthracene )DMBA Group. Gross of pouches presented tumor masses on the mucosa (A.

closed, B. opened). Microscopically, moderate (3) hyperplasia with moderate (3) dysplasia,

thickness of the epidermal layer (C. 100x, D. 200x, E. 400x). Severe (4) papilloma showed

cauliflower-like growth, elongated and protruded with necrosis from epidermis to form

papillary folding (F. 40x), and moderate (3) multicentric squamous cell carcinoma (SCC)

invaded into the submucosa (G. 100x) and increased mitoses (H. 200x, I. 400x) in the

epidermal epithelium of the pouch, H&E stain.

The effect of Physalin A on Gross and histopathological findings

of hamster’s pouch in DMBA Group

(6)

31 E

Gross and histopathological findings of hamster’s pouch in DMBA + PA 50 mg/kg Group.

Gross of pouches presented tumor masses on the mucosa (A. closed, B. opened, animal

code. 202). Microscopically, slight (2) hyperplasia with moderate (3) dysplasia, thickness

of the epidermal layer (C. 100x, D. 200x, E. 400x). Moderate (3) papilloma showed

cauliflower-like growth, elongated and protruded with necrosis from epidermis to form

papillary folding (F. 40x), and no (0) multicentric squamous cell carcinoma (SCC)

invaded into the submucosa (G. 100x) but increased mitoses (H. 200x, I. 400x) in the

epidermal epithelium of the pouch (animal code. 207), H&E stain.

The effect of Physalin A on Gross and histopathological findings

of hamster’s pouch in DMBA Group

32

Physalin A in Hamster pouch tumor expression

Ind

ex

sc

ale

of le

sio

n

s

0

1

2

3

4

Hyperplasia Dysplasia Papilloma Invasive carcinoma

DMBA - + + + +

PA mg/kg 0 0 2.5 5.0 7.5

Degrees of lesions

(1= minimal; 2=slight; 3=moderate; 4=severe)

33

AIF: apoptosis-inducing factor MMP-3 : matrix metalloproteinase-3 GRB2 : growth factor receptor-bound protein Comet assay DNA damage p53 CyclinA/B1 Wee1 G2/M phase arrest PA migration invasion metastasis

Epithelial-like Mesenchymal-like

MMP-2 GRB2 α-smooth muscle Actin E-cadherin Apoptosis active cleaved-caspase-3 4 0

Fas

cleaved-caspase-8

CAD: caspase activated Dnase ICAD: inhibtor CAD MMP : mitochondrial membrane potential DNA nuclear AIF DNA fragmentation DNA ladder formation CAD inhibitor inactive active DAPI stain DNA condensation cleaved-caspase-9 active AIF Ca2+

mitochondri

a

Ca2+ MMP (∆Ψm) Bcl-2 Bax Bid

Summary 1

34 Second part

Anti-inflammtory research of PA

by Ya-Sing Hsiau

Undergraduate student study

from 2011/3 to 2012/6

35

36 PA significantly decreased the NO production and

shown low cell cytotoxicity in

RAW

264.8 cells,

but the its mechanism still does not understand.

Cell Viability and the Effect of Physalin A on

LPS-induced NO Production in Macrophages

(7)

37 Inflammation Pathway

TLR4

mediators: NO PGs cytokines: IL-1β, IL-6,

TNF-α

Inflammation Pathway

y

TLR4 pathway (

Upstream

)

CD14, MD2, MyD88

y

MAPK pathway

JNK, ERK, p38…

y

IkK, IkB pathway

p-IkK, p-IkB…

y

NF-κB pathway

p50, p65

y

Transcription proteins

(Down

stream):

COX-2, iNOS, MMPs

y

mediators: NO, PGs...

y

cytokines:

IL-1

β, IL-6, TNF-α ...

TLR4

COX-2, iNOS, MMPs

38 y

In this study, we want to

evaluation the

anti-inflammatory effects of

physalin A.

y

In vitro:

using

lipopolysaccharide (LPS)

-stimulated mouse

macrophage

RAW264.7 cells

y

In vivo:

using λ-

carrageenan

(Carr)-induced hind mouse

paw edema model.

Aim

mediators: NO PGs cytokines: IL-1β, IL-6,

TNF-α COX-2, iNOS, MMPs

39 METHODS

In vitro

RAW

cell

MTT assay NO assay ELISA NO Ils TNF Western PCR mediators: NO PGs cytokines: IL-1b, IL-6,

TNF-a

In vivo

Mice

Carrageen

an-induced

Paw

Edema

NO assay ELISA NO Ils TNF Western

RESULTS

40

41 Inhibition of LPS-induced iNOS and

COX-2 Protein by Physalin A

Physalin A treatment could down-regulation of iNOS

and COX-2 proteins expression in RAW 264.7 cells.

42 Inhibition of LPS-induced NF-κB

Proteins by Physalin A

Down-regulation of p65 proteins, respectively, after the treatment

with physalin A at 2 µM compared with the LPS-alone.

(8)

43 Physalin A Inhibits the levels of I

ΚK ,

I

ΚB Inflammatory Proteins

IKB IK B pro te in ex pre s s ion( %c on tr ol) 0 20 40 60 80 100 120 140 160 180 PAEA(μg/ml) - 0.5 1 2 4 -LPS(100μg/ml) - + + + + + + Indomethasin(10μM) - - - + pERK R el at ive pr ot ei n expr essi on (f o ld of LPS t reat ed gr oup) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 med ACE- 0 ACE-25 ACE-50 ACE-75 ACE-100 # * *

pERK

ERK

pJNK R el a ti ve pr ot ei n expr essi on (f o ld of LPS t rea te d gr oup) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 med ACE- 0 ACE-25 ACE-50 ACE-75 ACE-100 * # * * *

pJNK

JNK

pp38

p38

β-actin

Effects of Physalin A on the LPS-stimulated

Activation of Mitogen-activated Protein Kinases

(MAPKs)

These results suggest that phosphorylation of MAPKs may be

involved in the inhibitory effect of physalin A on LPS-stimulated

NF-κB binding in RAW 264.7 cells.

45

MMP-9 Pe rc en ta g e ( % ) 0 20 40 60 80 100 120 control PA 0.375 PA 0.75 PA 1.5 PA 3.1 PA 6.25 MMP-9

P

er

ce

n

ta

ge

(%

)

0 20 40 60 80 100 120 control PA 0.375 PA 0.75 PA 1.5 PA 3.1 PA 6.25

Physalin A-Reduced MMP-2 & MMP-9

expression in zymography and western blotting

Time (hrs) 0 1 2 3 4 5 Δ v ( m l) 0.00 0.02 0.04 0.06 0.08 0.10 Carr

Carr + Indo 10 mg/kg (i.p.) Carr + PAEA 2.5 mg /kg (i.p.) Carr + PAEA 5.0 mg /kg (i.p.) Carr + PAEA 10 mg /kg (i.p.)