1. The Kallikrein-Kinin Cascade and Kinin receptors on Mesothelioma cells
Supervisors: Professor Kanti Bhoola
Odette Shaw
Cadet: Angeline Ng

2. Introduction Formation of kinins Activation of kinin receptors
Kallikrein-kininogen-kinin cascade
Kinin
Kininogen
Kallikrein

4. Kinins Bradykinin Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe DesArg9-Bradykinin
Lys-Bradykinin (Kallidin)
Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe
Lys-DesArg10-Bradykinin
Kininase I
-Arg 9
Kininase I
-Arg 10

6. Kinin Receptors B1 : induced by inflammation
B2 : constitutively expressed
B2: Bradykinin
B1: DesArg9-Bradykinin
B2/B1:Lys-Bradykinin (Kallidin)
B1:Lys-DesArg10-Bradykinin

7. Kinins stimulate: Activity of matrix metalloproteases
break down tissue matrix proteins
 metastasis
Gαq pathway cell proliferation

8. Kinins stimulate:
release of vascular endothelial growth factor  formation of new blood vessels
Release of prostaglandins and leukotrienes  increase vascular permeability

9. Tissue Kallikrein & Plasma Kallikrein
Expressed in human cancers of the lung and pleura
-hydrolyze macromolecules in
extracellular matrix metastasis
-formation of new blood vessels

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

11. Hypothesis 1
The cellular distribution of kinin B1 and B2 receptors differ in cultured NO36 mesothelioma cells

12. 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 months
Appearance:
-Thick, stellate shape cells with vacuoles

13. Aim 1
Determine the expression of B1 and B2 receptors on the human mesothelioma cell line: NO36

14. Y P Immunocytochemistry Y DAB DAB Antibodies studied:
-TproK, TK, PPK, PK11, LK, B1 and B2 P Y 2◦ Y 1◦
DAB
DAB
3.3’-diaminobenzidine R

15. Universal Rabbit IGg- Negative control Primary antibody omitted
Neutrophil controls
Positive control
Primary antibody
Universal Rabbit IGg- Negative control
Negative control
Primary antibody omitted
For each primary antibdy-100% neutrophil labelling was used as the positive control. Negative controls are indicated above.

16. TproK
100
200
400
600

18. TK 50
100
200
400

19. PPK
100
200
400
800

20. PK11 50
100
100
400
200

21. LK 50
100
200
400

22. B1
100
200
400
800
100
100
200
400
800

23. B2
100
200
400
800 50
100
200
400

24. Discussion
We are the first to assess the expression of kinin B1 and B2 receptors, kininogens and kallikreins in cultured mesothelioma cells

25. Kinin B1 and B2 Receptors Expression of B1> B2 on NO36 cells
antagonists that target B1 or both receptors could have therapeutic value
Other studies have shown that bradykinin and desArg9bradykinin act on B2 and B1 receptors respectively to increase cell proliferation and angiogenesis in a number of cancers (ie brain, lung)

26. Kallikreins and Kininogens
Peripheral : TK, PK11, LK
Whole cell: TproK, PPK
Conclusion:
TK, PK11, LK, TproK, PPK are present on NO36 cells
The Kinin-Kallikrein-inflammatory cascade is involved in mesothelioma
Potential for therapeutic intervention

27. Hypothesis 2
The monomeric and dimeric kinin receptor antagonists differ in the cellular effects on cultured NO36 mesothelioma cells

28. Aim 2
Compare cellular effects of monomeric and dimeric kinin receptor antagonists in
NO36 mesothelioma cells

29. Kinin Receptor Antagonists
Monomeric Antagonists
-B B9958
-B1/B2 B9430
Dimeric Antagonist
-B1/B2 B9870

30. Kinin Receptor Antagonists
Monomeric Gαq - inhibits Ca signal
Dimeric Gαq -inhibits Ca signal
Gα12,13 -induces apoptosis
-kills small cell lung
carcinoma cells

31. Antagonist/Agonist Study
Used flat-bottom 96 well plates
Each well had 10,000 cells
Once cells adhered to the bottom, media was aspirated and wells were treated with:
Agonists: BK, des-Arg9BK
Antagonists: B9958, B9430, B9870

32. Yellow tetrazolium salt Insoluble Formazancrystal
MTT Assay
Yellow tetrazolium salt
Reduced by mitochondria
Insoluble Formazancrystal
Detergent
Spectrophotometer
No. of viable cells
Solubilized Formazan
& cell lysis

33. Agonist Results

34. Agonist Results

35. Antagonist Results

36. Discussion After 24hrs, BK and dBK caused the cell numbers to decrease
Possible explanation: Non-specific toxicity.
-[BK] and [dBK] may be too high for
mesothelioma cells

37. Kinin Receptor Agonists
BK and dBK cause cell proliferation in small cell and non-small cell lung cancer
Perhaps they work by a different mechanism in mesothelioma.
ie stimulating angiogenesis
 promoting metastases

38. Kinin Receptor Agonists
After 48 hrs, BK and dBK caused no significant change in cell numbers
Possible explanations:
1.Reversible competitive protease inhibitors were added to each well to prevent the breakdown of BK and dBK.
After 48 hr, more proteases may have been synthesized by the cells  break down BK and dBK
2. B1 and B2 receptors were desensitized due to the high agonist concentrations

39. Kinin Receptor Antagonists
We are the first to assess the cellular effects of the antagonists B9958, B9430 & B9870 on cultured mesothelioma cells
The antagonists caused no significant change in cell numbers
consistent with Odette Shaw’s results on adenocarcinoma and squamous cell carcinoma cells.

40. Kinin Receptor Antagonists
Other studies have shown that these antagonists work in small cell and non-small cell lung cancer, and prostate cancer at the concentrations and time-points we used
But mesothelioma and non-small cell lung cancer respond to different treatment regimens
 they have different properties
Doses and time-points used may be unsuitable for mesothelioma cells

41. Further Research
Test distribution of B1 and B2 receptors on different cultured mesothelioma cell lines and normal mesothelial cell lines compare
Test desArg9BK, BK & the antagonists at different concentrations and time-points

42. References
Bhoola KD, Figueroa CD, Worthy K. Bioregulation of Kinins: Kallikreins, Kininogens, and kininases. Pharmacol Rev, 1992;44(1):1-80
Campbell DJ. Towards understanding the kallikrein-kinin system: insights from measurement of kinin peptides. Braz J Med Biol Res ;33(6):665-77
Stewart JM. Bradykinin Antagonists as Anti-Cancer Agents. Current Pharm Design. 2003;9:

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