Abstract
Imatinib is an effective therapy for chronic phase CML, but patients may became irresponsive due to the development of resistance, caused by amplification of the BCR-ABL genomic locus or by point mutations within the kinase domain of BCR-ABL, which prevents drug binding. Novel dual SRC/ABL kinase inhibitors with higher potency against native and imatinib-resistant mutants of BCR-ABL have substantial clinical utility, but at least one mutation remains resistant to any kinase inhibitor (T315I). Thus, the search for alternative drugs effective in CML is still cogent. Treatment of CML cells with histone deacetylase inhibitors (HDIs) of the class of hydroxamic acid analogues promotes proteasomal degradation of Bcr-Abl, associated with apoptosis, in synergy with imatinib. We evaluated whether HDIs of other classes, namely short chain fatty acids like butyrates and valproic acid, could exert the same effects and we intended to dissect the determining molecular mechanisms.
The human CML cell lines K562, KBM, LAMA-84 S and LAMA-84 R and primary imatinib-resistant CML-BC cells were grown in the presence of valproic acid (VPA) at the escalating doses 0.2 mM to 2mM or the mannose ester of butyric acid D1 (0.2–1mM) for 24 and 48 hrs. Apoptosis was induced in a time and dose dependent manner by VPA and D1 (annexin V test and flow cytometric analysis after propidium iodide uptake). Imatinib was synergistic with both HDIs in inducing apoptosis and cell proliferation arrest (MTT-assay). VPA and D1 were able to induce after 48 hrs of incubation a significant decrease in the number of copies of BCR-ABL determined by real time-PCR, paralleled by a substantial decrease in Bcr-Abl protein expression, shown in western blots of total cell lysates from CML cells. This decrease in the expression of protein kinase could account for the synergy with imatinib, but also for the reversal of resistance in mutated Bcr-Abl CML cells and is consistent with what previously observed. We also analysed the expression of Hsp-90 (protein chaperone of Bcr-Abl) and found it quantitatively unmodified but hyperacetylated by the treatment with both HDIs. As little is known of the ability of short chain fatty acids to induce acetylation of non-histone proteins, we compared the acetylated proteome of CML cells treated and not treated with HDIs, alone and in combination with imatinib, by 2D western blot versus a pan-acetylated antibody, followed by MALDI-TOF mass spectrometry for protein identification.
22 proteins were positively identified with a high degree of confidence, with the majority of these being cytoplasmic. At least two chaperone proteins were identified as target of acetylation after VPA and D1 treatment of CML cells, other targets were proteins involved in the synthesis and stability of RNA. Phosphorylation of proteins, evaluated by 2D western blot, was not significantly affected by HDIs.
Short chain fatty acids are indeed not the most potent HDIs, but have been used successfully in clinical trials. Our observations contribute to the dissection of proteome modifications by HDIs and may help extrapolate the molecular effects of different HDIs on CML cells so to improve their use as single drugs or in combination with imatinib or new SRC/ABL inhibitors.
Anticonvulsant valproic acid and other short-chain fatty acids as novel anticancer therapeutics: Possibilities and challenges
