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Blood, 1 December 2000, Vol. 96, No. 12, pp. 4001-4002
CORRESPONDENCE
To the editor:
Signaling mechanism in the induction of apoptosis by thrombin
in human tumor cells
We have read with great interest the recent article by Zain et
al.1 We have made similar observations regarding the
dual effect of thrombin in human tumor cells2 and would
like to note the following. The dual effects of thrombin (ie, inducing proliferation at
concentrations less than 0.5 U/mL and apoptosis at concentrations of at
least 0.5 U/mL) are effective on a wide variety of tumors. We have
observed these effects on all the human tumor cell lines that we have
examined (HeLa, U937, Jurkat, Daudi, CEM, Raji, HL60, Caco2, KSY-1,
K562, MCF-7, MDA-231, 1301, CA46, BL41, etc). It should be noted that
similar dual effects of thrombin have been observed on human neurons.
But the concentration required to induce apoptosis in these cells is
80- to 200-fold higher than that required in the tumor
cells.3,4 Zain et al1 did not mention these effects of thrombin on neurons. The normal human peripheral blood mononuclear cells (PBMC) are
relatively resistant to the proapoptotic effects of thrombin. The thrombin-induced apoptosis in these tumor cells occurs
without consistent changes in the expression of bcl-2, p53, or p21 proteins. It is highly unlikely that all the above tumor cell lines that we
tested and found susceptible to the thrombin-induced apoptosis express
PAR-1. It may suggest the involvement of receptors/molecules other than
PAR-1. Interestingly, we were also unable to observe apoptosis when the
tumor cells were incubated with the PAR-1 activating hexapeptide
(SFLLRN) at high concentrations, which could be due to the degradation
by proteases as suggested by Zain et al.1 In an attempt to learn about the signaling pathways involved in
the thrombin-induced apoptosis of the tumor cells, we incubated the
U937 tumor cells with thrombin with or without some known inhibitors of
cell signaling. As shown in Figure 1,
genistein, H-7, wortmanin, cytochalasin D, and DEVD-CHO significantly
prevented the growth inhibitory effects of thrombin. This suggests the
involvement of caspases, protein kinase C, PI3-K, unspecified tyrosine
kinases, and an intact cytoskeleton for the thrombin-induced
apoptosis in the human tumor cells. Interestingly, similar observations were made regarding the signaling pathways involved in the protective and proapototic effects of thrombin on neurons.3,4
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| Figure 1.
Effect of signal transduction inhibitors on the
thrombin-induced apoptosis.
The human tumor cells (U937) were incubated in triplicate in the wells
of a 96-well microculture plate (1 × 104 cells/well)
with or without the presence of thrombin (0.5 U/mL), and/or the
indicated inhibitors for 16 hours. The microcultures were pulsed with
0.037 MBq (1 µCi) of 3H-thymidine for 8 hours, and the
3H-thymidine uptake was determined using liquid
scintillation counting. Each bar represents average CPM ± SE from
triplicate microcultures. The star on the bar indicates that the
inhibitor was able to significantly (P  .05; Student
t test) block the thrombin-induced inhibition of
proliferation/apoptosis. The letters A-I on the x-axis indicate the
inhibitors used in the microcultures: (A) no thrombin; (B) 0.5 U/mL
thrombin; (C) 0.5 µmol/L herbimycin A (an inhibitor of phospholipase
D, tyrosine kinases, and anti-CD3 antibody mediated apoptosis); (D) 100 µmol/L genistein (a broad spectrum inhibitor of protein tyrosine
kinases); (E) 10 µmol/L H-7 (a potent inhibitor of PKC); (F) 100 µmol/L HA-1004 (an inhibitor of CaM kinase II, myosin light chain
kinase, and PKA); (G) 1 µmol/L wortmanin (an inhibitor of PI3-kinase,
and phospholipase D); (H) 0.5 µg/mL cytochalasin D (an inhibitor of
function of the cytoskeletal protein actin); and (I) 0.3 µM DEVD-CHO
(a cell permeable inhibitor of caspases 3, 6, 7, 8, and 10).
Microcultures C-I were incubated in the presence of thrombin (0.5 U/mL). All these inhibitors were from Calbiochem (San Diego,
CA).
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The relative resistance of normal human cells to the thrombin-induced
apoptosis, as compared to the tumor cells, suggests that thrombin or
its mimetic peptides may be potentially useful as anticancer agents and
in the future may lead to novel thrombin-based anticancer therapies.
Rasheed Ahmad, Jose Menezes, and Ali Ahmad
Laboratory of Immunovirology Research Center Sainte Justine
Hospital Montreal, Quebec, Canada
References
1.
Zain J, Huang Y-Q, Feng XS, et al.
Concentration-dependent dual effect of thrombin on impaired growth/apoptosis or mitogenesis in tumor cells.
Blood.
2000;95:3133-3138[Abstract/Free Full Text].
2.
Ahmad R, Knafo L, Xu J, et al.
Thrombin induces apoptosis in human tumor cells.
Intl J Cancer.
2000;87:707-715[Medline]
[Order article via Infotrieve].
3.
Donovan FM, Cunningham DD.
Signaling pathways involved in thrombin-induced cell protection.
J Biol Chem.
1998;273:12746-12752[Abstract/Free Full Text].
4.
Simirnov IV, Zhang SX, Citron BA, et al.
Thrombin is an extracellular signal that activates intracellular death protease pathways inducing apoptosis in model motor neurons.
J Neurobiol.
1998;36:64-80[Medline]
[Order article via Infotrieve].
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