Combination of Bortezomib and Mitotic Inhibitors Down-Modulate Bcr-Abl and Efficiently Eliminates Tyrosine-Kinase Inhibitor Sensitive and Resistant Bcr-Abl-Positive Leukemic Cells

Emergence of resistance to Tyrosine-Kinase Inhibitors (TKIs), such as imatinib, dasatinib and nilotinib, in Chronic Myelogenous Leukemia (CML) demands new therapeutic strategies. We and others have previously established bortezomib, a selective proteasome inhibitor, as an important potential treatment in CML. Here we show that the combined regimens of bortezomib with mitotic inhibitors, such as the microtubule-stabilizing agent Paclitaxel and the PLK1 inhibitor BI2536, efficiently kill TKIs-resistant and -sensitive Bcr-Abl-positive leukemic cells. Combined treatment activates caspases 8, 9 and 3, which correlate with caspase-induced PARP cleavage. These effects are associated with a marked increase in activation of the stress-related MAP kinases p38MAPK and JNK. Interestingly, combined treatment induces a marked decrease in the total and phosphorylated Bcr-Abl protein levels, and inhibits signaling pathways downstream of Bcr-Abl: downregulation of STAT3 and STAT5 phosphorylation and/or total levels and a decrease in phosphorylation of the Bcr-Abl-associated proteins CrkL and Lyn. Moreover, we found that other mitotic inhibitors (Vincristine and Docetaxel), in combination with bortezomib, also suppress the Bcr-Abl-induced pro-survival signals and result in caspase 3 activation. These results open novel possibilities for the treatment of Bcr-Abl-positive leukemias, especially in the imatinib, dasatinib and nilotinib-resistant CML cases

A novel caspase 8 selective small molecule potentiates TRAIL-induced cell death

Recombinant soluble TRAIL and agonistic antibodies against TRAIL receptors (DR4 and DR5) are currently being created for clinical cancer therapy, due to their selective killing of cancer cells and high safety characteristics. However, resistance to TRAIL and other targeted therapies is an important issue facing current cancer research field. An attractive strategy to sensitize resistant malignancies to TRAIL-induced cell death is the design of small molecules that target and promote caspase 8 activation. For the first time, we describe the discovery and characterization of a small molecule that directly binds caspase 8 and enhances its activation when combined with TRAIL, but not alone. The molecule was identified through an in silico chemical screen for compounds with affinity for the caspase 8 homodimer’s interface. The compound was experimentally validated to directly bind caspase 8, and to promote caspase 8 activation and cell death in single living cells or population of cells, upon TRAIL stimulation. Our approach is a proof-of-concept strategy leading to the discovery of a novel small molecule that not only stimulates TRAIL-induced apoptosis in cancer cells, but may also provide insights into the structure-function relationship of caspase 8 homodimers as putative targets in cancer

Modulation of TRAIL resistance in colon carcinoma cells: Different contributions of DR4 and DR5

<p>Abstract</p> <p>Background</p> <p>rhTRAIL is a therapeutic agent, derived from the TRAIL cytokine, which induces apoptosis in cancer cells by activating the membrane death receptors 4 and 5 (DR4 and DR5). Here, we investigated each receptor's contribution to rhTRAIL sensitivity and rhTRAIL resistance. We assessed whether agonistic DR4 or DR5 antibodies could be used to circumvent rhTRAIL resistance, alone or in combination with various chemotherapies.</p> <p>Methods</p> <p>Our study was performed in an isogenic model comprised of the SW948 human colon carcinoma cell line and its rhTRAIL resistant sub-line SW948-TR. Effects of rhTRAIL and agonistic DR4/DR5 antibodies on cell viability were measured using MTT assays and identification of morphological changes characteristic of apoptosis, after acridine orange staining. Sensitivity to the different death receptor ligands was stimulated using pretreatment with the cytokine IFN-gamma and the proteasome inhibitor MG-132. To investigate the mechanisms underlying the changes in rhTRAIL sensitivity, alterations in expression levels of targets of interest were measured by Western blot analysis. Co-immunoprecipitation was used to determine the composition of the death-inducing signalling complex at the cell membrane.</p> <p>Results</p> <p>SW948 cells were sensitive to all three of the DR-targeting agents tested, although the agonistic DR5 antibody induced only weak caspase 8 cleavage and limited apoptosis. Surprisingly, agonistic DR4 and DR5 antibodies induced equivalent DISC formation and caspase 8 cleavage at the level of their individual receptors, suggesting impairment of further caspase 8 processing upon DR5 stimulation. SW948-TR cells were cross-resistant to all DR-targeting agents as a result of decreased caspase 8 expression levels. Caspase 8 protein expression was restored by MG-132 and IFN-gamma pretreatment, which also re-established sensitivity to rhTRAIL and agonistic DR4 antibody in SW948-TR. Surprisingly, MG-132 but not IFN-gamma could also increase DR5-mediated apoptosis in SW948-TR.</p> <p>Conclusions</p> <p>These results highlight a critical difference between DR4- and DR5-mediated apoptotic signaling modulation, with possible implications for future combinatorial regimens.</p

A Peroxidase Peroxiredoxin 1-Specific Redox Regulation of the Novel FOXO3 microRNA Target let-7

Abstract Precision in redox signaling is attained through posttranslational protein modifications such as oxidation of protein thiols. The peroxidase peroxiredoxin 1 (PRDX1) regulates signal transduction through changes in thiol oxidation of its cysteines. We demonstrate here that PRDX1 is a binding partner for the tumor suppressive transcription factor FOXO3 that directly regulates the FOXO3 stress response. Heightened oxidative stress evokes formation of disulfide-bound heterotrimers linking dimeric PRDX1 to monomeric FOXO3. Absence of PRDX1 enhances FOXO3 nuclear localization and transcription that are dependent on the presence of Cys31 or Cys150 within FOXO3. Notably, FOXO3-T32 phosphorylation is constitutively enhanced in these mutants, but nuclear translocation of mutant FOXO3 is restored with PI3K inhibition. Here we show that on H2O2 exposure, transcription of tumor suppressive miRNAs let-7b and let-7c is regulated by FOXO3 or PRDX1 expression levels and that let-7c is a novel target for FOXO3. Conjointly, inhibition of let-7 microRNAs increases let-7-phenotypes in PRDX1-deficient breast cancer cells. Altogether, these data ascertain the existence of an H2O2-sensitive PRDX1-FOXO3 signaling axis that fine tunes FOXO3 activity toward the transcription of gene targets in response to oxidative stress. Antioxid. Redox Signal. 28, 62–77

Playing the DISC:Turning on TRAIL death receptor-mediated apoptosis in cancer

Formation of the pro-apoptotic death-inducing signaling complex (DISC) can be initiated in cancer cells via binding of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to its two pro-apoptotic receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2. Primary components of the DISC are trimerized TRAIL-R1/-R2, FADD, caspase 8 and caspase 10. The anti-apoptotic protein FLIP can also be recruited to the DISC to replace caspase 8 and form an inactive complex. Caspase 8/10 processing at the DISC triggers the caspase cascade, which eventually leads to apoptotic cell death. Besides TRAIL, TRAIL-R1- or TRAIL-R2-selective variants of TRAIL and agonistic antibodies have been designed. These ligands are of interest as anti-cancer agents since they selectively kill tumor cells. To increase tumor sensitivity to TRAIL death receptor-mediated apoptosis and to overcome drug resistance, TRAIL receptor ligands have already been combined with various therapies in preclinical models. In this review, we discuss factors influencing the initial steps of the TRAIL apoptosis signaling pathway, focusing on mechanisms modulating DISC assembly and caspase activation at the DISC. These insights will direct rational design of drug combinations with TRAIL receptor ligands to maximize DISC signaling. (C) 2009 Elsevier B.V. All rights reserved

Bortezomib and paclitaxel combined treatment significantly downregulates phosphorylated Bcr-Abl levels, inhibiting the phosphorylation/activity of downstream STAT3/STAT5, CrkL and Lyn kinase-dependent pathways, in K562 & LAMA84 cell lines.

<p><b>A</b>. K562 leukemic cells were treated with 9nM bortezomib and 6nM paclitaxel for 48h, followed by detection of the phosphorylated levels of Bcr-Abl, and of the total and phosphorylated protein levels of STAT3, STAT5, CrkL and Lyn. The combined regimen significantly downregulates the phosphorylation of Bcr-Abl, CrkL and Lyn kinases, the total levels and phosphorylation of STAT5 and the phosphorylation of STAT3 transcription factors. <b>B</b>. LAMA84 leukemic cells were treated with 4nM bortezomib and 5nM paclitaxel for 48h, followed by detection of the phosphorylated levels of Bcr-Abl, and of the total and phosphorylated levels of STAT3, STAT5, CrkL and Lyn. The combined regimen significantly downregulates the phosphorylation of Bcr-Abl, CrkL and Lyn kinases, and the total levels and phosphorylation of STAT5. β-Actin was used as a loading control. One representative experiment from three separate experiments is shown.</p

Downregulation of active caspase 8 as a mechanism of acquired TRAIL resistance in mismatch repair-proficient colon carcinoma cell lines

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers the apoptotic cascade in various colon cancer cell lines after binding to the membrane receptors DR4 and DR5. However, not all cancer cell lines are sensitive to the therapeutic recombinant human TRAIL (rhTRAIL). To investigate the causes of TRAIL resistance in colon cancer cell lines, models have been developed, mostly in mismatch repair-deficient cells. These cells are prone to mutations in genes containing tandem repeat, including pro-apoptotic protein Bax. We therefore investigated the mechanism underlying TRAIL resistance acquisition in a mismatch repair-proficient colon carcinoma cell line. The TRAIL-resistant cell line SW948-TR was established from the TRAIL-sensitive cell line SW948 by continuous exposure to rhTRAIL, and exhibited 140-fold less sensitivity to rhTRAIL in a cell viability assay. Resistance was stable for over a year in the absence of rhTRAIL. Both cell lines had similar TRAIL receptor cell membrane expression levels. Treatment with the protein synthesis inhibitor cycloheximide sensitized SW948-TR to rhTRAIL-induced apoptosis, indicating that the functionality of the TRAIL receptors was maintained. In SW948-TR, procaspase 8 protein levels but not mRNA levels were notably lower than in SW948. Downregulation of c-FLIP with short interfering RNA (siRNA) sensitized SW948-TR cells to rhTRAIL while caspase 8 siRNA decreased rhTRAIL sensitivity in SW948, indicating the importance of the caspase 8/c-FLIP ratio. Proteasome inhibition with MG 132 did not restore basic procaspase 8 levels but stabilized cleaved caspase 8 in rhTRAIL-treated SW948-TR cells. Altogether, our results suggest that colon cancer cells can acquire rhTRAIL resistance by primarily reducing the basal procaspase 8/c-FLIP ratio and by increasing active caspase 8 degradation after rhTRAIL treatment. Proteasome inhibitors can effectively overcome acquired rhTRAIL resistance in mismatch repair-proficient colon cancer cells

Combined treatment of bortezomib and paclitaxel efficiently activates caspases and induces cell death in human leukemic Bcr-Abl-positive K562 and LAMA84 cell lines.

<p><b>A</b>. K562 leukemic cells were exposed to bortezomib (9nM) with or without paclitaxel (6nM) for 48h. The percentage of cell death was measured with an automated Trypan Blue exclusion method. The combination significantly increased the number of Trypan Blue-positive cells, compared with each drug used alone. The results represent the mean +/- standard deviations (SDs) of 4 measurements/condition for the representative Western blot experiment presented in (B). <b>B</b>. K562 leukemic cells were treated with 9nM bortezomib and 6nM paclitaxel for 48h, followed by detection of the cleaved fragments of caspase 3, and of PARP cleavage. The combined regimen induced significant cleavage of caspases 3, 8 & 9, and PARP, implying caspase activation. <b>C</b>. LAMA84 leukemic cells were exposed to bortezomib (4nM) with or without paclitaxel (5nM), for 48h. The percentage of cell death was measured with an automated Trypan Blue exclusion method. The combination significantly increased the number of Trypan Blue-positive cells, compared with each drug used alone. The results represent the mean +/- standard deviations (SDs) of 5 measurements/condition for the representative Western blot experiment presented in (D). <b>D</b>. LAMA84 leukemic cells were treated with 4nM bortezomib and 5nM paclitaxel for 48h, followed by detection of the cleaved fragments of caspase 3, caspase 8, caspase 9 and of PARP cleavage. The combined regimen significantly enhanced the cleavage of caspases 3, 8, 9 and PARP, suggesting caspase activation. At least 3 separate experiments were performed in each case. β-Actin was used as a loading control; “***” = p<0.0001; “**” = p<0.01;.</p

Bortezomib/paclitaxel combination induces downregulation of the total levels and phosphorylation of Bcr-Abl in TKIs-resistant K562-R, LAMA84-R and Baf3 Bcr-Abl T315I, displaying increased Bcr-Abl expression/activity and/or T315I mutation.

<p><b>A</b>. K562 (K562-S) and K562-R leukemic cells were treated with 9nM bortezomib and 6nM paclitaxel for 48h, followed by detection of the total levels and phosphorylation of Bcr-Abl. The combined regimen significantly decreases the phosphorylation of Bcr-Abl in both cell lines. <b>B</b>. LAMA84 (LAMA84-S) and LAMA84-R leukemic cells were treated with 4nM bortezomib and 5nM paclitaxel for 48h, followed by detection of the total levels and phosphorylation of Bcr-Abl. The combined regimen downregulates the total levels and phosphorylation of Bcr-Abl in LAMA84-R and phosphorylation of Bcr-Abl in LAMA84-S. <b>C</b>. Baf3 Bcr-Abl and Baf3 Bcr-Abl T315I cell lines were treated with 7nM bortezomib and 7nM paclitaxel for 48h, followed by detection of the total levels and phosphorylation of Bcr-Abl. The combined regimen decreases the phosphorylation of Bcr-Abl in both cell lines. β-Actin was used as a loading control. One representative experiment from several separate experiments is shown.</p