Haematologica 2003 - Supplements

357
Thalidomide protects endothelial cells from
doxorubicin-induced injury but alters cell morphology
Varsha Kaushal,Gur P Kaushal, Sonia Mlekaveri, Manish
Kohli, Louis Fink,Paulette Mehta
University of Arkansas for Medical Sciences and the Central
Arkansas Veterans Healthcare System, Little Rock, AR
Thalidomide has anti-angiogenesis activity and is effective in
many patients with multiple myeloma. It is however associated
with an increased of thrombosis, especially in patients who have
received prior anthracycline chemotherapy. Anti-angiogenesis
and thrombosis are both endothelial-related activities, and we
therefore speculated that the interaction of both agents on
endothelium could promote thrombogenic activity. We therefore
evaluated in vitro effects of thalidomide on intact cultured
endothelial cells, and on cultured endothelial cells injured by preincubation
with doxorubicin. Endothelial cells (human coronary
artery endothelial cells) were purchased from Clonetics
Corporation and grown in endothelial basal medium with 5%
heat-inactivated fetal bovine serum. Cells were treated with
varying doses of doxorubicin (l-2µ/L) for l to 48 hours and cells
were then harvested by scraping and centrifugation. Cell lysates,
supernatant, and extract were prepared. Cell viability was
measured by MTT assay. Caspase-3 activity was done by
measurement of fluorescent leaving group after cleavage at the
Asp residue using fluorescent spectrofluorometry. PAR-l receptor
assay was done using PAR-l antibody in an enhanced
chemiluminescence assay system. Immunostaining and
fluorescent microscopy were done on paraformaldehyde-fixed
cells on coverslips incubated with FITC-conjugated phalloidin
followed by nuclear staining with propidium iodide. Cells were
imaged using fluorescent microscopy at 400x magnification.
Results showed caspase-3 activity was increased within one hour
after doxorubicin treatment, continued to increase for up to 8
hours, and then stabilized. Doxorubicin caused a significant
dose-dependent increase in caspase-3 activity when used in
concentrations from 0 to 6µmol/L. Thalidomide alone did not
induce caspase-3 activity. Cell pretreated with doxorubicin
(6µmol/L) for 6 hours, followed by thalidomide incubation,
resulted in decreased caspase-3 activity. Doxorubicin caused a
progressive time-dependent decrease in cell viability from l to 48
hours (77% to 25%); whereas thalidomide treatment alone did not
alter cell viability. When endothelial cells were co-incubated
with doxorubicin and thalidomide, there was less decrease in
cell viability than when endothelial cells were treated with
doxorubicin alone (76 to 5l% vs 77 to 29% cell viability) .
Doxorubicin caused endothelial cell apoptosis within 24 hours,
and this was prevented by treatment with thalidomide (l0g/ml).
When thalidomide was incubated with cells exposed to
doxorubicin, there was immediate and marked formation of
neotubules and pro-angiogenesis. Neotubule and proangiogenesis
effects were not seen when thalidomide was
incubated with untreated endothelial cells. The neotubule
formation was related to thrombin receptor activation since
thrombin receptor activation occurred in doxorubicin-treated, but
not in untreated, endothelial cells, as measured by
immunoblotting using PAR-l antibody. These findings suggest
that thalidomide may be procoagulant and proangiogenic through
stimulation of thrombin receptors of doxorubicin-injured
endothelium and may explain the increased hypercoagulability in
patients treated with anthracycline chemotherapy followed by
thalidomide.
358
EFFECT OF ANTICOAGULATION ON DEVELOPMENT
AND RECURRENCE OF DEEP VEIN THROMBOSIS
(DVT) IN MULTIPLE MYELOMA PATIENTS TREATED
WITH CHEMOTHERAPY AND THALIDOMIDE (TOTAL
THERAPY II)
Maurizio Zangari, Bart Barlogie, Fariba Saghafifar, Paul
Eddlemon, Joth Jacobson, Choon-Kee Lee, Raymond
Thertulien, Giampaolo Talamo, Teri Thomas, Frits van
Rhee, Elias Anaissie, Athanasios Fassas, Louis Fink and
Guido Tricot
The Myeloma Institute for Research and Therapy (MIRT),
University of Arkansas for Medical Sciences, Little Rock, AR, USA;
Cancer Research And Biostatistics (CRAB), Fred Hutchinson
Cancer Center, Seattle, WA, USA; Central Arkansas Veteran
Healthcare System, John L. McClellan Memorial Veterans
Hospital, Little Rock, AR, USA.
As part of Total Therapy II, a group of newly diagnosed multiple
myeloma patients (n=256) were randomly assigned to identical
cytotoxic treatment with or without thalidomide at 400 mg per
day. The induction phase consisted of 4 cycles of combination
chemotherapy (VAD, DCEP, CAD with PBSC collection, DCEP)
followed by tandem transplant with melphalan 200mg/m2 and,
post-tandem transplant consolidation chemotherapy for one year,
and maintenance interferon. Deep vein thrombosis (DVT) was
significantly more frequent in the thalidomide group (HR: 4.5;
p