SIRT1 Activator 3 Potently Induces Apoptosis in Multiple Myeloma Cell Lines and in Primary Human Multiple Myeloma Cells.

Multiple myeloma (MM) is a malignant plasma cells disorder and as far as now remains an incurable disease so that new target therapies are necessary to improve survival of MM patients. Histone deacetylases (HDACs) are enzymes that deacetylate acetyl lysines in histones and various non-histone proteins. Class I and II HDACs have been identified as valid anticancer targets so that clinical studies on their inhibitors as new anticancer agents are ongoing. Much less is known about the consequences of inhibition and activation of the seven members of class III HDACs (sirtuins, SIRT1-7). Recent studies highlight the role of SIRT1 in stress response and cell survival. Increased levels of SIRT1 were observed in normal colon mucosa but not in advanced colon carcinomas. Moreover, SIRT1 activators have been shown to have anti-inflammatory property mediated by TNF-alpha.

We have investigated the anti-myeloma activity of SIRT1 activator 3 in RPMI8226 and U226 MM cell lines and in 4 primary human MM cells.

In this study RPMI8226 and U226 MM cell lines were used. Bone marrow samples of 3 MM patients were subjected to CD138 immunomagnetic purification for plasmacells enrichment. Peripheral plasmacells from a patients with plasmacell leukaemia dexametasone-melphalan resistant were also collected.

SIRT1 activator 3 was used at increasing dose of 50, 100 and 500 μM alone and in combination with 1 μM of dexametasone in MM cell lines and at the fixed dose of 500 μM in primary human plasmacells. Apoptosis has been assayed at 12h, 24h and 48h after treatment in RPMI8226 and U226 cells and at 24h in human plasmacells by flow cytometry evaluating annexin V marker. Western blot analysis was performed to assess the effect of SIRT1 activator 3 agent on NFkB activity (localization of p65 subunit), AKT, p-AKT, mTOR, p-mTOR, Src and p-Src.

The highest level of apoptosis was observed in RPMI8226 cells with SIRT1 activator 3 agent at the dosage of 500 μM at 24 h. (annexin V positivity 53%, P<0.05). U226 cells resulted sensitive in the same conditions ((annexin V positivity 41%, P<0.05).

SIRT1 activator 3 (500 μM) induced significant apoptosis at 24h (range 29-47%) in MM cells of all four patients. The highest level of apoptosis was observed in plasmacells of the patient with plasma cell leukaemia dexametasone-melphalan resistant (47% annexin V positivity)

Dexametasone do not significantly enhances SIRT1 activator 3 induced apoptosis in both MM cell lines and primary human MM cells.

Western blot analysis demonstrated strong reduction of AKT, mTOR and Src phosphorylation in RPMI8226 and U226 cells as early as 24h after exposure.

SIRT1 activator 3 induces significant cell death in MM cell lines and primary human myeloma cells in a dose-dependent manner. The mechanisms of SIRT1 activator 3 cytotoxicity are related to down-regulation of AKT, SRC and TOR phosphorylation. Together, these findings may give useful insights into a novel anti-myeloma therapy.

Guglielmelli:Celgene: Honoraria; Janssen Cilag: Honoraria. Saglio:Celgene: Honoraria; Novartis: Honoraria; Bristol Myers: Honoraria.