Abstract
Since a number of CML patients are resistant to Imatinib, additional molecular defects should be identified and targeted to improve the therapeutic strategies. A constitutive NF-kB activity has been demonstrated in several hematologic malignancies, therefore NF-kB blocking approaches have been introduced as antineoplastic strategies. The phosphorilation of IKB by IKK leads to IKB degradation by the proteasome, so freeing NF-kB to enter the nucleus and activate transcription. IKK may therefore represent an attractive target for molecular therapies. The aim of the study was to evaluate the effects of the IKK inhibitor PS1145 (Millenium) in CML cell lines and patients sensitive and resistant to Imatinib. K562 and KCl cells both sensitive (s) and resistant (r) to Imatinib and the BM cells collected from 13 CML patients were incubated with PS1145 10 μM, with Imatinib 1 μM and with the combination of the two drugs for 24 and 48 hrs. 11 out of 13 patients were in chronic phase, 1 in accelerate phase and 1 in blastic phase. 3 out of 11 were cytogenetic resistant, one was also hematologically resistant. The NF-kB activity was evaluated by ELISA method, the proliferation rate by MTT assay, apoptosis by FACS. Immunofluorescence was use to identify the NF-kB localization. Moreover colony growth was evaluated in control and treated samples. PS1145 was able to reduce the DNA binding activity of NF-kB of 90% and 85% respect to the control in K562 and KCL cells.This was confirmed by the prevailing cytoplasmatic localization of NF-kB after PS1145 incubation. In K562s and KCls treated with Imatinib, we observed a reduction of proliferation of 48% and 37% respectively. By contrast no effect on proliferation was observed in K562r and KClr. The incubation with PS1145 inhibited the proliferation of 38% and 15% in K562s and KCls, and of 22% and 28% in K562r and KClr. Interestingly in resistant cell lines the association of Imatinib and PS1145 induced a marked inhibition of proliferation of 87% in K562r and 82% in KClr. Similarly, PS1145 inhibited BM cells proliferation of 30% (range 11% to 65%). Imatinib incubation of BM cells from sensitive patients reduced the proliferation rate of 41% (range 29%–55%) but no effects were observed in resistant patients. In the three resistant patients the incubation with both compounds resulted in an increased block of proliferation respect to PS1145 alone with an inhibition of 42%, 49% and 58% after 24 hrs and 70%, 77% and 79% after 48 hrs. Imatinib plus PS1145 induced a significant increase of apoptosis from 7% to 69% in K562r and from 9 % to 71% in KClr. In agreement this association induced 48%, 52% and 39% of apoptotic cells in BM from the three resistant patients and a colony growth inhibition of 86%. Our data clearly demonstrated that the IKK inhibitor PS1145 is able to induce growth arrest and apoptosis in CML cell lines and BM cells. This effect is more sound in Imatinib resistant cells treated with the association of Imatinib and PS1145. Although the exact mechanism of action of PS1145 in resistant cells is at present a pure speculation, these data may suggest an intriguing mechanism to induce apoptosis in imatinib resistant cells based on imatinib resistance itself. The combination of Imatinib and the IKK inhibitor could therefore represent a valid approach for the treatment of CML patients resistant to Imatinib therapy.
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