The KallikreinKinin Cascade and Kinin receptors on Mesothelioma cells

Supervisors: Professor Kanti Bhoola Odette Shaw Cadet: Angeline Ng

Introduction
  

Formation of kinins Activation of kinin receptors Kallikrein-kininogen-kinin cascade
Kininogen Kini n

Kallikrei n

Kinins
•Bradykinin
1 2 3 6 7 8 Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe Arg •DesArg9-Bradykinin 4 5 Kininas eI 9

Kininas •Lys-Bradykinin (Kallidin) eI 1 2 3 4 5 6

7 8 9 Lys-Arg-Pro-Pro-Gly-Phe-Ser-ProArg Phe

10

•Lys-DesArg10- Bradykinin

Kinin Receptors

B2: Bradykinin B1: DesArg9-Bradykinin B2/B1:Lys-Bradykinin (Kallidin) B1:Lys-DesArg10 -Bradykinin

Kinin Receptor Antagonists
 Monomeric

Antagonists

-B1 -B1/B2

B9958 B9430

 Dimeric

Antagonist

-B1/B2

B9870

Kinin Receptor Antagonists

Monomeric

G αq

- inhibits Ca signal

Dimeric

Gαq -inhibits Ca signal Gα12,13 -induces apoptosis
-kills small cell lung carcinoma cells

Cancer

Tissue Kallikrein (hK1) and Plasma Kallikrein (hKB1)
Expressed in human cancers of the lung and pleura -TK and PK hydrolyze macromol in extracellular matrix metastasis -formation of new blood vessels


Kinins

Kinins are mitogenic peptides -regulate growth and proliferation of cancer cells -increase vascular perm

Mesothelioma
 

Mesothelium: lung pleura Grows over serosal surfaces and inter-lobular fissures of the lung

Iannella & Mummolo 2007

Mesothelioma

Metastases: lymph nodes, liver, adrenal gland, kidney, bone Latent period: 15-40 years Mortality: 75% within 1 yr of diagnosis Cause: asbestos fibers, contaminated water, coal tar, ionising radiation

  

Hypothesis 1

The cellular distribution and expression of kinin B1 and B2 receptors differ in cultured NO36 mesothelioma cells

Human mesothelioma cell line

NO36:

-derived from pleural effusions of untreated mesothelioma patients -malignancy confirmed by cytology -history of crocidolite asbestos exposure

Primary culture for 11-25 months Appearance:
-Thick, stellate shape cells with vacuoles

Aim 1

Determine expression of B1 and B2 receptors on the human mesothelioma cell line: NO36 Method: Dako EnVision ICC
2◦
1◦

DAB

3.3’-diaminobenzidine

R

Antibodies
Anti-tissue prokallikrein (TproK) Polyclonal rabbit against CAP 11 (synthetic peptide) that covers the aminoterminal residues of TproK. Polyclonal rabbit against against TK, IgG isotype

Anti-tissue kallikrein (TK)

Antibodies
Anti-plasma prekallikrein Polyclonal rabbit against (PPK) human PPK

Anti-plasma kallikrein (PK11)

Monoclonal rabbit against light chain of human plasma kallikrein, subtype IgG2aк (epitope class D), prepared from lyophilised ascites fluid

Antibodies
Anti-B1 receptor Polyclonal rabbit against 4 fusion proteins of various domains of 27-52 residues from human bradykinin B1 receptor

Anti-B2 receptor

Polyclonal rabbit against synthetic peptides of the N-terminal and transmembrane loop regions of rat bradykinin B2 receptor All antibodies were reconstituted and diluted with
0.01M phosphate-buffered saline containing 1% IgGfree bovine serum albumin and 0.2% NaN3. Note: Odette will get back to me about PK11 and LK info

Neutrophil controls
Positive control Primary antibody Universal Rabbit IGgNegative control Negative control Primary antibody omitted

For each primary antibdy-100% neutrophil labelling was used as the positive control. Negative controls are indicated above.

TproK

100

200

400

600

TproK
120 100 80 60 40 20 0 100 200 400 600 Dilutions

% cells brown

TK

5 0

100

200

40 0

TK
120 100 80 60 40 20 0 50 100 200 400 Dilutions

% cells brown

PPK

10 0

200

400

800

PPK
100 % cells brown 80 60 40 20 0 100 200 400 800 Dilutions

PK11

50

10 100 0

200

400

PK11
120 100 80 60 40 20 0 50 100 200 400 Dilutions

% cells brown

LK

50

100

200

40 0

LK
50 % cells brown 40 30 20 10 0 50 100 200 400 Dilutions

B1
200

100 100

100

200

400

800

400

80 0

B1

B1

120 100 80 60 40 20 0 100 200 400 800 Dilutions

% cells brown

B2
100 200

400

50

800

100

200

40 0

100 % cells brown 80 60 40 20 0 50 100

B2

200

400

Dilutions

Hypothesis 2

The monomeric and dimeric kinin receptor antagonists differ in the cellular effects on cultured NO36 mesothelioma cells

Aim 2

Compare cellular effects of monomeric and dimeric kinin receptor antagonists in NO36 mesothelioma cells

Kinin Receptor Antagonists

Monomeric Antagonists
B9958 B9430

-B1 -B1/B2

Kinin Receptor Antagonists
 Dimeric

Antagonist

-B1/B2

B9870

-get details from manuscript

MTT Assay
Yellow tetrazoli um salt

Reduced by mitochondria

Insolubl e Formaz ancryst al

Detergent

No. of viabl e cells

Spectrophotom eter

Solubilize & cell d lysis Formazan

Antagonist Results
NO36 24 hours
1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 B9870 4uM B9870 2uM B9870 1uM B9958 4uM B9958 2uM Media

NO36 48hr plate 1
**
Absorbance
1.75 1.50 1.25 1.00 0.75 0.50 0.25

**

Absorbance

B9958 1uM

B9430 4uM

B9430 2uM

B9430 1uM

0.00 B9870 4uM B9870 2uM B9870 1uM B9958 2uM B9958 1uM B9430 2uM B9430 1uM Media B9958 4uM B9430 4uM

Treatment

Treatment

This is a rough representation. The title will be standardised to just 24hr or 48hr and the colour scheme on the

Agonist Results
NO36 24hr plate 2
1.5

NO36 48hr plate 2
1.5

** **

**

Absorbance

Absorbance

1.0

1.0

0.5

0.5

0.0

0.0
Media BK 10 BK 1 dBK 10 dBK 1

Media

BK 10

BK 1

dBK 10

dBK 1

Treatment

Treatment

The graphs will be standardised to look like the 2 graphs in the previous slide with titles being 24hr or 48hr

Discussion

Thank you
Prof Kanti Bhoola  Dr Neil Misso  Odette Shaw  Prof Phil Thompson  Dr Paul Rigby  Dr Lajos Gere  Prof John Stewart

References

Campbell DJ. Towards understanding the kallikrein-kinin system: insights from measurement of kinin peptides. Braz J Med Biol Res. 2000 ;33(6):66577 Stewart JM. Bardykinin Antagonists as Anti-Cancer Agents. Current Pharm Design. 2003;9:2036-2042

Kininases

Factor XIIa

Pre-kallikrein Peptidases that cleave kinins

llidi
Lys -

Campbell 2000

Bacterial LPS, damaged basement memb

Receptors
B1 receptor: Normally absent. Upregulated by: inflammatory mediators, ischaemia, bacterial products,transplant rejection Agonist: [des-Arg] kinin.

B2 receptor: constitutively expressed Mediates acute nociceptive,inflammatory,

vasoactive responses Agonist: Bradykinin

Cliff Robinson (www.che.udel.edu/cobre/gpcr.

Effects
Inflammation Arteriole dilate (NO release), increase perm  edema,lower BP Stim cytokine release from monocytes  attract leukocytes

Bradykinin (agonist) Kallidin (agonist) 1st generation

Arg-Pro-Pro-Gly-Phe-Ser-Pro-PheArg

Lys-Arg-Pro-Pro-Gly-Phe-Ser-ProPhe-Arg

Antibodies
  

residues (Ala1-Arg7) of (TproK) followed by the 1st 3 residues (Ile8 to Val10) of the mature enzyme, with an additional Cys residue at the N-terminus Cys-Ala-Pro-Pro-Ile-Gln-Ser-Arg-Ile-Val-Gly Cys: residue added for coupling reactions Ala-Pro-Pro-Ile-Gln-Ser-Arg= activation peptide of the zymogen form of tissue kallikrein Ile-Val-Gly: aminoterminus of activated kallikrein

PK11: monoclonal antibody to human plasma kallikrein light chain; epitope class D; (IgG2aк), lyophilized ascites HMW kininogen: polyclonal antibody IgG Plasma kallikrein: antiserum to human plasma kallikrein raised in rabbit; lyophilized antiserum, reconstituted with 0.01M PBS containing 1% IgG free BSA and 0.2% NaN3 tProk: Antibody to synthetic peptide made of the 1st 7 N-terminal