The Proteasome Inhibitor Bortezomib and Histone Deacetylase Inhibitor SAHA Sinergistically Inhibit Proliferation and Induce Apoptosis of Megakaryoblastic MO7-e Cells.

PS-341 (Bortezomib) is a novel dipeptide boronic acid proteasome inhibitor with in vitro and in vivo antitumor activity that induces apoptosis in different human cancer cell lines.

Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has been reported to induce apoptosis of leukemic cells by increasing the cyclin-dependent kinase inhibitor p21 or generating reactive oxygen species (ROS).

Co-exposure of BCR/ABL-positive cells both sensitive and resistant to imatinib to these compounds has been previously reported resulting in an increased apoptotic rate.

To extend this observation, we examined the effect of treatment with bortezomib or/and SAHA of a megakaryoblastic cell line (MO7-e). Cell proliferation, ROS production, cell cycle progression, induction of apoptosis and differentiation has been investigated.

Bortezomib was shown to retain NF-kB in the cytoplasm and inhibit cell growth (IC50=20nM), in a dose/time-dependent way. This anti-proliferative activity resulted to be lineage-specific, because other leukemic cell lines were unaffected by the bortezomib treatment.

Moreover, bortezomib in MO7-e cells increased ROS production and induced a significant pro-apoptotic effect (50% vs 5% in control).

Finally, cell cycle was blocked in the G2 phase and bortezomib was able to down-regulate WT1 expression, gene that could play a relevant prognostic role in myeloproliferative disorders. Moreover, any significant effect on cell differentiation was found.

SAHA also resulted able to inactivate NF-kB and to inhibit cell proliferation, at 1.5 uM. It did not increase significantly ROS production, blocked cell cycle in the G1 phase and down-regulated WT1 expression (10 fold minus than bortezomib). Neverthelles, SAHA also did not induce differentiation of megakaryoblatic cells.

Co-exposure of this cell line to minimally toxic concentrations of bortezomib (5 nM) and SAHA (0.5 uM), resulted in a significant increase of anti-proliferative (50% of growth inhibition vs 15% with bortezomib and 10% with SAHA alone) and pro-apoptotic effect (45% vs 20% of bortezomib and 15% of SAHA alone).

Interestingly, immunocitochemistry assays detecting the NF-kB p65 subunit showed that the co-exposure to bortezomib and SAHA resulted in a minor NF-kB inactivation than that achieved with single compounds.

This finding was confirmed by the pre-incubation of MO7-e cells with SAHA in respect of the pre-incubation with bortezomib or the simultaneously addition of the two drugs. In fact, pre-incubating megakaryoblasts with SAHA, the anti-proliferative effect of bortezomib significantly decreased.

In conclusion, this study supports the association of a proteasome with a histone deacetylase inhibitor, in a time-sequence-related way, especially in chronic myeloproliferative disorders where a spontaneous NF-kB activation and a WT1 over-expression have been reported.