Valproic acid sensitizes chronic lymphocytic leukemia cells to apoptosis and restores the balance between pro- and antiapoptotic proteins

Abstract

Chronic lymphocytic leukemia (CLL) is one of the most common leukemias in adults in the developed world. Despite significant advances in the treatment of cancer, CLL remains incurable. The main feature of the disease is the generation of circulating B-cells with prolonged survival caused by aberrant apoptosis. In this study, we observe that valproic acid (VPA), a well-established histone deacetylase (HDAC) inhibitor, mediates apoptosis in CLL cells ex vivo through caspase activation via both the extrinsic and the intrinsic apoptosis pathways, as indicated by the activation of the caspase proteins 8 and 9, and cleavage of the proapoptotic protein BID. The Bcl-2/Bax ratio was decreased as a consequence of decreased bcl-2 mRNA levels in response to treatment with VPA. With the results presented in this study, we have identified the HDAC inhibitor VPA as restoring the apoptotic pathways in CLL cells and thus their ability to undergo apoptosis.

Figures

Figure 1

VPA-mediated cell death in CLL cells. Mononuclear cells isolated from peripheral blood of CLL patients were treated as indicated and analyzed by FACS. (A) Dot blot analyses are shown for FSC/SSC and for annexin V/PI for a representative patient analyzed after 48 h of culture. (B) Cell viability was monitored for 30 patients over 7 days at different concentrations of VPA. The graphs display the decrease in viable cells (annexin V−/PI−) under VPA treatment normalized to untreated cells. VPA-treated CLL cells show increased histone acetylation. (C) PBMCs from a CLL patient were left untreated (−) or were cultivated in the presence of different VPA concentrations (0.1, 1, 3 mM) for 48 h. Histone extracts were subjected to Western blot analysis using acetylation-specific antibodies for histones H4 and H3. Equal loading of gels was documented with anti-actin antibody.

Figure 2

Impact of VPA on the viability of PBMCs from healthy donors. (A) PBMCs from 5 healthy donors and from CLL patients were isolated, and spontaneous apoptosis was assessed by FACS. The diagram shows the relative amounts of viable cells (annexin V−/PI−) monitored over 7 days. (B) The graphs display the decrease in viable cells (annexin V−/PI−) from healthy donors monitored over 7 days of treatment in the presence of absence of different VPA concentrations normalized to untreated cells. (C) VPA-dependent apoptosis at 1 mM VPA was compared in PBMCs from CLL patients and healthy donors. The diagram presents the relative amounts of viable cells (annexin V−/PI−) normalized to untreated cells. (D) The table shows the significances (P values) determined for VPA-dependent apoptosis induction in CLL cells versus PBMCs from healthy donors. The viability of CD3+ cells (E) and CD19+ cells (F) from healthy donors during VPA treatment was compared with similarly treated CLL cells.

Figure 3

VPA-induced apoptosis is caspase dependent. (A) PBMCs from a representative CLL patient were cultured either in the presence or absence (−) of VPA (0.1, 0.5, 1, 2, 3 mM) for 3 days. Levels of caspases 8 and 9 were assessed from cellular lysates by Western blot. Equal loading of gels was documented with anti-actin antibody. Relative intensities of protein bands for p20 (activated caspase 8) (B) pro-caspase 8 (C), and pro-caspase 9 (D) were quantified with the TotalLab software and normalized to actin. Quantifications include data from at least 5 independent experiments.

Figure 4

VPA modifies the expression of pro- and antiapoptotic CLL proteins. mRNA expression was measured by real-time PCR in 10 patients after 24 h of culture in the presence or absence of VPA. (A) bcl-2 expression of treated cells is shown in relation to untreated control cells. Student’s t test values reveal significant values for all tested VPA concentrations: 0.1 mM (P < 0.05), 1 mM (P < 0.001), and 3 mM (P < 0.001). (B) Gene expression values were normalized to gapdh, and the bcl-2/bax ratio was calculated. (C) bcl-2, bax, and bak levels were analyzed by Western blotting of total cellular extracts of CLL cells from 1 representative patient after culture for 3 days. (D) VPA-dependent apoptosis is accompanied by cleavage of BID. CLL cellular extracts were obtained after a 3-day culture in the presence or absence of VPA (2 representative patients are shown). BID and truncated BID (t-BID) were detected by Western blotting.

Figure 5

VPA-mediated apoptosis in CLL cells involves both apical caspases. Primary CLL cells were cultured for 48 h in the presence or absence of 1, 2, or 3 mM VPA, the inhibitor solvent DMSO, caspase 8 inhibitor (c8), caspase 9 inhibitor (c9), or a combination of caspase inhibitors and 2 mM VPA. (A) Caspase 3 and 7 activities were analyzed and are presented relative to the untreated control (n = 5). (B) Western blot analysis of total cellular extracts was done for caspase 8 and 9 and BID; equal loading was ensured by reprobing with the actin antibody.

Figure 6

A model for the valproate mediated apoptosis sensitization in CLL cells. In order to futher analyze our observation that valproate promotes cell death in cultured CLL cells, we analyzed the single steps of apoptosis induction and indentified an activation of both classical apoptosis pathways. Following experiments indicated that CASPASE 8, the activator caspase of the extrinsic pathway, was activated first. The cleavage of BID provided a connection to the intrinsic pathway and the activation of CASPASE 9. This is further supported by the downregulation of the antiapoptotic protein Bcl-2, which leads to increased sensitivity towards the induction of apoptosis.