ABSTRACT
AIMS: The authors' aim was to conduct a proof-of-principle study to test whether c-Jun N-terminal kinase (JNK) phosphorylation and Noxa induction occur in peripheral blood chronic lymphocytic leukaemia (CLL) cells in patients receiving a vincristine infusion. METHODS: Patients with CLL received 2 mg vincristine by a 5-min intravenous infusion. Blood samples were collected at baseline and up to 6 h after the vincristine infusion, and assayed for JNK activation, Noxa induction and vincristine plasma concentrations. RESULTS: Ex vivo treated peripheral CLL cells activated JNK in response to 10-100 nM vincristine in 6 h. Noxa protein expression, while variable, was also observed over this time frame. In CLL patients, vincristine infusion led to rapid (<1 h) JNK phosphorylation in peripheral blood CLL cells which was sustained for at least 4-6 h after the vincristine infusion. Noxa protein expression was not observed in response to vincristine infusion. CONCLUSIONS: This study confirmed that vincristine can activate JNK but not induce Noxa in CLL cells in vivo. The results suggest that novel JNK-dependent drug combinations with vincristine warrant further investigation.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , JNK Mitogen-Activated Protein Kinases/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , Vincristine/pharmacology , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/pharmacokinetics , Enzyme Activation/drug effects , Humans , Infusions, Intravenous , JNK Mitogen-Activated Protein Kinases/blood , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Lymphocytes/drug effects , Lymphocytes/enzymology , Phosphorylation , Proto-Oncogene Proteins c-bcl-2/metabolism , Vincristine/administration & dosage , Vincristine/pharmacokineticsABSTRACT
This review summarizes the current state of scientific understanding of the apoptosis pathway, with a focus on the proteins involved in the pathway, their interactions and functions. This forms the rationale for detailing the preclinical and clinical pharmacology of drugs that modulate the pivotal proteins in this pathway, with emphasis on drugs that are furthest advanced in clinical development as anticancer agents. There is a focus on describing drugs that modulate three of the most promising targets in the apoptosis pathway, namely antibodies that bind and activate the death receptors, small molecules that inhibit the anti-apoptotic Bcl-2 family proteins, and small molecules and antisense oligonucleotides that inactivate the inhibitors of apoptosis, all of which drive the equilibrium of the apoptotic pathway towards apoptosis. These structurally different yet functionally related groups of drugs represent a promising novel approach to anticancer therapeutics whether used as monotherapy or in combination with either classical cytotoxic or other molecularly targeted anticancer agents.
Subject(s)
Antineoplastic Agents/therapeutic use , Apoptosis Regulatory Proteins/metabolism , Apoptosis/drug effects , Neoplasms/drug therapy , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Apoptosis Regulatory Proteins/antagonists & inhibitors , Clinical Trials as Topic , Humans , Neoplasms/metabolism , Neoplasms/pathology , Receptors, Death Domain/agonists , Treatment OutcomeABSTRACT
Proteins of the BCL2 family provide a survival mechanism in many human malignancies, including chronic lymphocytic leukemia (CLL). The BCL2 inhibitor ABT-263 (navitoclax) is active in clinical trials for lymphoid malignancies, yet resistance is expected on the basis of preclinical models. We recently showed that vinblastine can dramatically sensitize several leukemia cell lines to ABT-737 (the experimental congener of ABT-263). The goal of these experiments was to determine the impact of vinblastine on ABT-737 sensitivity in CLL cells isolated from peripheral blood and to define the underlying mechanism. Freshly isolated CLL cells from 35 patients, as well as normal lymphocytes and platelets, were incubated with various microtubule-disrupting agents plus ABT-737 to assess sensitivity to the single agents and the combination. ABT-737 and vinblastine displayed a range of sensitivity as single agents, and vinblastine markedly sensitized all CLL samples to ABT-737 within six hours. Vinblastine potently induced the proapoptotic protein PMAIP1 (NOXA) in both time- and dose-dependent manner and this was required for the observed apoptosis. Combretastatin A4, which dissociates microtubules by binding to a different site, had the same effect, confirming that interaction of these agents with microtubules is the initial target. Similarly, vincristine and vinorelbine induced NOXA and enhanced CLL sensitivity to ABT-737. Furthermore, vinblastine plus ABT-737 overcame stroma-mediated resistance to ABT-737 alone. Apoptosis was induced with clinically achievable concentrations with no additional toxicity to normal lymphocytes or platelets. These results suggest that vinca alkaloids may improve the clinical efficacy of ABT-263 in patients with CLL.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Biphenyl Compounds/pharmacology , Drug Resistance, Neoplasm , Gene Expression Regulation, Leukemic/drug effects , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Nitrophenols/pharmacology , Proto-Oncogene Proteins c-bcl-2/genetics , Sulfonamides/pharmacology , Vinblastine/pharmacology , Antineoplastic Agents, Phytogenic/toxicity , Apoptosis/drug effects , Apoptosis/genetics , Biphenyl Compounds/toxicity , Blood Platelets/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/genetics , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Lymphocytes/drug effects , Nitrophenols/toxicity , Piperazines/pharmacology , Piperazines/toxicity , Prognosis , Proto-Oncogene Proteins c-bcl-2/metabolism , Stromal Cells/metabolism , Sulfonamides/toxicity , Tubulin Modulators/pharmacology , Tubulin Modulators/toxicity , Vinblastine/toxicityABSTRACT
Naturally occurring nitroalkene fatty acids (NAs) derived from oleic (NO(2)-OA) and linoleic (NO(2)-LA) acids mediate a variety of cellular responses. We examined the signaling pathways involved in NA activation of Nrf2/ARE-dependent versus PPARγ/PPRE-dependent transcription in human MCF7 breast cancer cells. Additionally, we compared the relative potencies of NO(2)-OA and NO(2)-LA in activating these two transcriptional programs. Here it is demonstrated that, in addition to the direct adduct formation of NA with the Nrf2 inhibitory protein, Keap1, shown by others, NA activation of Nrf2/ARE-mediated transcription results from increased nuclear Nrf2 levels and depends upon activation of the PI3K/AKT and PKC, but not ERK and JNK MAPK, signaling pathways. Examination of the relationship between NA stimulation of the Nrf2/ARE versus PPARγ/PPRE transcriptional programs revealed concentration-dependent activation of distinct signaling pathways that were readily distinguished by selective attenuation of Nrf2/ARE-dependent, but not PPARγ-dependent, transcription by inhibitors of PI3K and PKC. Moreover, measurable, statistically significant activation of PPARγ/PPRE-dependent transcription occurred at nanomolar concentrations of NAs-the 12-NO(2) isomer of NO(2)-LA showing the most potent activity-whereas significant activation of Nrf2/ARE-dependent transcription occurred at much higher NA concentrations (≥3 µM) with the NO(2)-OA isomers the most potent. These findings have implications for the physiological roles of NAs, suggesting that, at concentrations likely to be encountered in vivo, their direct activation of PPARγ transcription will dominate over their electrophilic activation of Nrf2 antioxidant/protective responses.
Subject(s)
Linoleic Acids/pharmacology , NF-E2-Related Factor 2/metabolism , Oleic Acids/pharmacology , PPAR gamma/metabolism , Response Elements , Signal Transduction , Transcription, Genetic , Alkenes/chemistry , Cell Line, Tumor , Humans , Linoleic Acids/chemistry , MAP Kinase Kinase 4/metabolism , NF-E2-Related Factor 2/genetics , Oleic Acids/chemistry , PPAR gamma/geneticsABSTRACT
The efficacy of many chemotherapeutic agents can be attenuated by expression of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Mcl-1. Flavopiridol and dinaciclib are cyclin-dependent kinase 7 and 9 inhibitors that transcriptionally inhibit expression of Mcl-1. We have investigated the ability of flavopiridol and dinaciclib to sensitize a panel of leukemia cell lines to vinblastine and paclitaxel. Both drugs acutely sensitized most of the leukemia lines to vinblastine, with 100% apoptosis in 4 h. Furthermore, dinaciclib sensitized freshly isolated chronic lymphocytic leukemia cells to vinblastine. This rapid induction of apoptosis was attributed to vinblastine-mediated activation of JNK because (a) flavopiridol and dinaciclib failed to induce apoptosis when combined with non-JNK activating concentrations of vinblastine; (b) JNK inhibitors suppressed JNK activity and prevented apoptosis; (c) flavopiridol did not potentiate apoptosis induced by paclitaxel which does not activate JNK in these cells; and (d) Jurkat cells failed to activate JNK in response to vinblastine and were not sensitive to combinations of vinblastine and flavopiridol or dinaciclib. The rapid induction of apoptosis by this combination in multiple cell systems but not in normal lymphocytes provides justification for performing a clinical trial to assess the efficacy in patients.
Subject(s)
Apoptosis/drug effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Flavonoids/pharmacology , Piperidines/pharmacology , Pyridinium Compounds/pharmacology , Vinblastine/pharmacology , Anthracenes/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Cyclic N-Oxides , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/metabolism , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Dose-Response Relationship, Drug , Drug Synergism , HL-60 Cells , Humans , Immunoblotting , Indolizines , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , Jurkat Cells , Myeloid Cell Leukemia Sequence 1 Protein , Paclitaxel/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , U937 Cells , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
BH3 mimetics are small molecules designed or discovered to mimic the binding of BH3-only proteins to the hydrophobic groove of antiapoptotic BCL2 proteins. The selectivity of these molecules for BCL2, BCL-X(L), or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to inhibit antiapoptotic proteins in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 as assessed by displacement of BAD and BIM from BCL2. The other six BH3 mimetics activate the endoplasmic reticulum stress response inducing ATF4, ATF3, and NOXA, which can then bind to and inhibit MCL1. In most cancer cells, inhibition of one antiapoptotic protein does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. Because MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. Our findings highlight a novel signaling pathway through which many BH3 mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies.
Subject(s)
Biphenyl Compounds/pharmacology , Endoplasmic Reticulum Stress/drug effects , Nitrophenols/pharmacology , Peptide Fragments/pharmacology , Peptidomimetics/pharmacology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins/pharmacology , Sulfonamides/pharmacology , Up-Regulation/drug effects , Activating Transcription Factor 3/genetics , Activating Transcription Factor 3/metabolism , Activating Transcription Factor 4/genetics , Activating Transcription Factor 4/metabolism , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Endoplasmic Reticulum Stress/genetics , Humans , K562 Cells , Membrane Proteins/genetics , Membrane Proteins/metabolism , Myeloid Cell Leukemia Sequence 1 Protein , Piperazines/pharmacology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Up-Regulation/genetics , bcl-Associated Death Protein/genetics , bcl-Associated Death Protein/metabolismABSTRACT
The naturally occurring nitroalkenes, nitrolinoleic (NO(2)-LA) and nitrooleic (NO(2)-OA) acids, are among the most potent endogenous ligand activators of PPARgamma-dependent transcription. In order to understand mechanisms that regulate cellular response to these nitroalkenes, we previously demonstrated that glutathione conjugation of NO(2)-LA and MRP1-mediated efflux of the conjugates were associated with significant attenuation of PPARgamma activation by this nitroalkene [(2006) Biochemistry 45, 7889-7896]. Here we show that NO(2)-OA activation of PPARgamma is similarly affected by nonenzymatic conjugation and MRP1-mediated efflux. Moreover, the roles of glutathione S-transferases (GSTs) in the glutathione conjugation and bioactivities of NO(2)-LA and NO(2)-OA were investigated. While none of the GST isozymes tested (GSTA1-1, A4-4, M1a-1a, and P1a-1a) enhanced the rate of glutathione conjugation, expression of GSTA1-1, M1a-1a, or P1a-1a in MCF7 cells significantly reduced the magnitude of PPARgamma-dependent reporter gene transcription in response to NO(2)-LA and NO(2)-OA treatment, with GSTP1a-1a expression mediating the most potent inhibition of PPARgamma. Although these GSTs failed to catalyze nitroalkene conjugation with glutathione, the nitroalkenes were found to associate avidly with all four GST isozymes as indicated by their ability to inhibit GST activity with K(i)'s in the nanomolar range. Treatment of purified GSTP1a-1a with excess NO(2)-LA and NO(2)-OA resulted in the formation of covalent adducts between GSTP1a monomers and nitroalkenes, although separate experiments indicated that such covalent bond formation was not necessary for avid GST-nitroalkene interactions. These results suggest that GSTs can inhibit the activation of transcription by nitroalkenes via noncatalytic sequestration of these ligands, and their glutathione conjugates, away from their nuclear target, PPARgamma.
Subject(s)
Alkenes/pharmacology , Fatty Acids/pharmacology , Glutathione Transferase/antagonists & inhibitors , Nitro Compounds/pharmacology , PPAR gamma/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Glutathione Transferase/chemistry , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Humans , Isoenzymes/chemistry , Isoenzymes/pharmacology , Kinetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Solubility , Spectrophotometry, UltravioletABSTRACT
Recent data has shown that nitrolinoleic acid (LNO(2)), an electrophilic derivative of linoleic acid, has several important bioactivities including antiinflammatory, antiplatelet, vasorelaxation, and-as a novel potent ligand of PPARgamma-transcription regulating activities. Moreover, LNO(2) is formed in abundance in vivo at levels sufficient to mediate these bioactivities. In order to investigate the role of glutathione conjugation and MRP1-mediated efflux in the regulation of PPARgamma-dependent LNO(2) signaling, regioisomers of LNO(2) were synthesized and characterized. Analysis by 1D and 2D (1)H and (13)C NMR revealed that the LNO(2) preparation consisted of four, rather than two, nitrated regioisomers in approximately equal abundance. At physiologic pH and intracellular glutathione levels, LNO(2) was rapidly and quantitatively converted to glutathione conjugates (LNO(2)-SG) via Michael addition. MRP1 mediated efficient ATP-dependent transport of LNO(2)-SG. Using a PPRE-containing reporter gene transiently transfected into MRP-poor MCF7/WT cells, we verified that the LNO(2) mixture was a potent activator of PPARgamma-dependent transcription. However, expression of MRP1 in the stably transduced MCF7 derivative, MCF7/MRP1-10, resulted in strong inhibition of LNO(2)-induced transcription activation. Taken together, these results suggest that glutathione conjugation and MRP1-mediated conjugate transport can attenuate LNO(2) bioactivity and thereby play important roles in the regulation of cellular signaling by LNO(2).