Visit our Expo - Redox and Inflammation signaling 2012

Survey on in vitro cell death induction of multiple myeloma cells upon valproic acid
treatment.
Chantal Schwartz, Valérie Palissot, René N.H.C. Brons and Guy Berchem.
Laboratoire d’Hémato-Cancérologie Expérimentale, CRP-Santé, 84, Val Fleury, L-1526
Luxembourg. E-mail :berchem.guy@chl.lu
Multiple myeloma (MM) is, despite the emergence of new treatments in recent years, still a
lethal disease in 2005. Over the last several years, significant insights into the dysregulation
of various signal transduction pathways of MM have emerged and have evolved the
development of new agents. Histone deacetylase (HDAC) inhibitors as valproic acid (VPA)
are compounds that inhibit HDAC enzymes that, in conjunction with histone acetylases,
regulate the acetylation state of histones and, by extension, the conformational status of
chromatin. VPA is clinically known to be used in treatment of different types of epileptic
diseases. The inhibitory functions of VPA on class I HDAC in combination with its moderate
secondary effects in therapy was recently exploited (Gottlincher M, 2004). VPA has been
described as inducer of apoptosis and differenciation in various cancer types and leukaemia.
In our study, effects of cell death have been studied in cells harvested and purified from
routine bone marrow aspirates of several patients with multiple myeloma, as well on the
myeloma cell lines OPM2, RPMI and U266 treated with a physiological range of VPA (1-2
mM). Using an XTT based cytotoxicity assay, we observe significant in vitro toxicity already
starting at doses of 1 mM for 24h in the cell lines OPM2 (60% viability) and RPMI (80%
viability), as well as on CD138+ selected MM cells from 2 patients. The cell line U266 was
more resistant (90% viability). Incubating in presence of ZVAD, a pan-caspase inhibitor, had
a partial protective effect, but did completely inhibit the cytotoxicity of VPA on OPM2 and
RPMI, which indicates that cell death through VPA is not only triggerd by the apoptotic
pathway. This was confirmed by flow cytometric analysis with PI/Annexin V staining, where
the cell lines U266 and OPM2 did not show apoptosis features, nor in DNA fragmentation
assays. However Western blot analysis of OPM2 and RPMI with antibodies against caspase 9
and 3 showed an accumulation of their cleaved forms within a time course of 72h of VPA
treatment, indicating that cell death induced by VPA in MM is not completely independent of
caspase activity.
In summary, these data suggest that VPA has a cytotoxic effect on MM cells, and partially but
not primarily induces apoptosis on these cells. Further experiments exploring other possible
mechanisms of cell death will be conducted.
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