The Kallikrein-

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

•Lys-Bradykinin (Kallidin)
Kininas
eI
1 2 3 4 5 6 10
7 8 9 -
Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-
Phe Arg
•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 B9958
-B1/B2 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 Polyclonal rabbit against CAP
prokallikrein (TproK) 11 (synthetic peptide) that
covers the aminoterminal
residues of TproK.

Anti-tissue kallikrein Polyclonal rabbit against

(TK) against TK,
IgG isotype
 Antibodies
Anti-plasma prekallikrein Polyclonal rabbit against
(PPK) human PPK

Anti-plasma kallikrein Monoclonal rabbit against

(PK11) 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% IgG-
free bovine serum albumin and 0.2% NaN3. Note:
Odette will get back to me about PK11 and LK info
 Neutrophil controls
Positive control Universal Negative
Primary Rabbit IGg- control
antibody Negative Primary
control 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
% cells brown

100
80
60
40
20
0
100 200 400 600
Dilutions
 TK

5 100
0

200 40
0
 TK

120
% cells brown

100
80
60
40
20
0
50 100 200 400
Dilutions
 PPK

10 200
0

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
% cells brown

100
80
60
40
20
0
50 100 200 400
Dilutions
 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
% cells brown

100
80
60
40
20
0
100 200 400 800
Dilutions
 B2
100 200

50 100
400 800

200 40
0
 B2
100
% cells brown

80
60
40
20
0
50 100 200 400
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
-B1 B9958
-B1/B2 B9430
Kinin Receptor Antagonists
 Dimeric Antagonist
-B1/B2 B9870
-get details from manuscript
 MTT Assay

Yellow Insolubl
tetrazoli Reduced by e
um salt mitochondria Formaz
ancryst
al

Detergent

No.
Spectrophotom Solubilize & cell
of
eter d
viabl lysis
e Formazan
cells
 Antagonist Results
NO36 24 hours NO36 48hr plate 1
1.2 ** 1.75
1.1 **
1.0 1.50
0.9
1.25
Absorbance

Absorbance
0.8
0.7
1.00
0.6
0.5 0.75
0.4
0.3 0.50
0.2
0.1 0.25
0.0
0.00
B9870 4uM
B9870 2uM
B9870 1uM
B9958 4uM
B9958 2uM
B9958 1uM
B9430 4uM
B9430 2uM
B9430 1uM
Media

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 NO36 48hr plate 2
1.5 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):665-
77
 Stewart JM. Bardykinin Antagonists
as Anti-Cancer Agents. Current
Pharm Design. 2003;9:2036-2042
 Kininases
Factor XIIa

 Peptidases that cleave kinins

Pre-kallikrein

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 Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-
Arg
(agonist)

Kallidin (agonist) Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-

Phe-Arg

1st generation
 Antibodies
 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
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