Haematologica 2003 - Supplements

We attached the trivalent arsenical, phenylarsonous acid, to the
free thiol of glutathione, a GlyCysGlu tripeptide, to produce 4-
(N-(S-glutathionylacetyl)amino) phenylarsenoxide (GSAO). The
water-solubility of GSAO restricts its entry into cells, which we
thought might improve the efficacy/toxicity ratio. The
corresponding pentavalent arsenical, 4-(N-(Sglutathionylacetyl)amino)phenylarsonic
acid (GSAA), was used
as the control for GSAO. The effects of As2O3, GSAO and
GSAA on the viability of three cultured human myeloma cell
lines (ARH77, RPMI 8226 and U266) was measured. Apoptosis
of myeloma cells was assessed by flow cytometry, using binding
of Annexin V-FITC and 7-amino-actinomycin D (7AAD).
Annexin V binds to anionic phospholipids exposed early in
apoptosis, while 7AAD staining of nuclear DNA indicates a
subsequent loss of membrane integrity. Consistent with previous
reports, 0.01mM As2O3 induced apoptosis of all three cell lines,
which was augmented in the presence of 0.1mM ascorbic acid.
Incubation with GSAO for 24h also induced greater than 90%
apoptosis at 0.5mM in the ARH77 and U266 cell lines, and at a
concentration of 0.1mM for RPMI-8226 cells. In both ARH77
and U266 cells, the addition of 0.1mM ascorbic acid did not
increase the effect of GSAO. The control compound, GSAA, did
not induce apoptosis in the cell lines. These data indicate that
GSAO is toxic for myeloma cells, although it is not as potent as
As2O3. The lack of augmentation by ascorbic acid suggests that
GSAO’s mechanism of action is different to that of As2O3. There
were no signs or symptoms of toxicity from thrice weekly S.C.
administration of 10 mg/kg GSAO to mice for 22 weeks. We now
plan to compare the efficacy versus toxicity of GSAO and As2O3
in a mouse model of myeloma.
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Proliferation Studies and Biochemical Analysis of
PS341 in Cells from Primary Plasma Cell Leukemia
Patients.
Atanasio Pandiella1, Azucena Esparís-Ogando1, Norma C.
Gutierrez2, Jesús F. San Miguel2
1Centro de Investigación del Cáncer, Universidad de Salamanca-
CSIC, Salamanca,Spain; 2Hematology, Hospital universitario de
Salamanca, Salamanca, Spain.
Primary plasma cell leukemia(PCL) is a rare disroder
characterized by increased numbers of circulating plasma cells,
common organ and tissue infiltration and very poor prognosis.
Novel treatment strategies are required in order to improve the
outcome of these patients. We report here the in vitro effect of the
proteasome inhibitor PS341 on fresh PC obtained from patients
with primary PCL. The effect of PS341 was compared with that
of four other drugs: dexamethasone, the Erk pathway inhibitor
PD98059, the Akt pathway inhibitor LY294002, and the
farnesyltransferase inhibitor FPTIII. The PC were characterized
by immunophenotyping and molecular cytogenetics showing
complex structural and numerical chromosomal changes. In both
patients PS341 caused a potent inhibition of PC growth as
measured by an MTT assay: 10nM PS341 induced a reduction of
cell viability to 20% (control untreated cells being considered as
100%) with an IC50 around 2 nM. This inhibition was dose
dependent. In patient 1, but not in patient 2, the FPTIII caused a
marked effect on cell viability (50%
reduction in M-protein following cycle 1 of therapy with one
patient achieving near complete remission, Seven patients had
stabilization of their disease process. 1 of the stable disease
patients progressed in the midst of the second cycle. Preliminary
results suggest the combination is active in this group of relapsed
myeloma patients. This initial response could be partly explained
by the use of the Dex. Historically, single agent Dex responses
are not durable; longer follow up will elucidate the effect of
Trisenox’s® role in sensitizing myeloma cells, to Dex as
reflected in response and durability.
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