RESUMO
Vinca alkaloids have been approved as anticancer drugs for more than 50 years. They have been classified as cytotoxic chemotherapy drugs that act during cellular mitosis, enabling them to target fast growing cancer cells. With the evolution of cancer drug development there has been a shift towards new "targeted" therapies to avoid the side effects and general toxicities of "cytotoxic chemotherapies" such as the vinca alkaloids. Due to their original classification, many have overlooked the fact that vinca alkaloids, taxanes and related drugs do have a specific molecular target: tubulin. They continue to be some of the most effective anticancer drugs, perhaps because their actions upon the microtubule network extend far beyond the ability to halt cells in mitosis, and include the induction of apoptosis at all phases of the cell cycle. In this review, we highlight the numerous cellular consequences of disrupting microtubule dynamics, expanding the textbook knowledge of microtubule destabilising agents and providing novel opportunities for their use in cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Moduladores de Tubulina/farmacologia , Animais , Antimitóticos/efeitos adversos , Antimitóticos/farmacologia , Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Humanos , Microtúbulos/efeitos dos fármacos , Terapia de Alvo Molecular , Neoplasias/patologia , Moduladores de Tubulina/efeitos adversos , Alcaloides de Vinca/efeitos adversos , Alcaloides de Vinca/farmacologiaRESUMO
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.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leucemia Linfocítica Crônica de Células B/enzimologia , Vincristina/farmacologia , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/farmacocinética , Ativação Enzimática/efeitos dos fármacos , Humanos , Infusões Intravenosas , Proteínas Quinases JNK Ativadas por Mitógeno/sangue , Leucemia Linfocítica Crônica de Células B/sangue , Linfócitos/efeitos dos fármacos , Linfócitos/enzimologia , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Vincristina/administração & dosagem , Vincristina/farmacocinéticaRESUMO
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.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Ensaios Clínicos como Assunto , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Receptores de Morte Celular/agonistas , Resultado do TratamentoRESUMO
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.
Assuntos
Compostos de Bifenilo/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Nitrofenóis/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptidomiméticos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas/farmacologia , Sulfonamidas/farmacologia , Regulação para Cima/efeitos dos fármacos , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Estresse do Retículo Endoplasmático/genética , Humanos , Células K562 , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Regulação para Cima/genética , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
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.
Assuntos
Ácidos Linoleicos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Ácidos Oleicos/farmacologia , PPAR gama/metabolismo , Elementos de Resposta , Transdução de Sinais , Transcrição Gênica , Alcenos/química , Linhagem Celular Tumoral , Humanos , Ácidos Linoleicos/química , MAP Quinase Quinase 4/metabolismo , Fator 2 Relacionado a NF-E2/genética , Ácidos Oleicos/química , PPAR gama/genéticaRESUMO
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.
Assuntos
Alcenos/farmacologia , Ácidos Graxos/farmacologia , Glutationa Transferase/antagonistas & inibidores , Nitrocompostos/farmacologia , PPAR gama/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/farmacologia , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Espectrofotometria UltravioletaRESUMO
Targeting anti-apoptotic BCL2 family proteins has become an attractive therapeutic strategy for many cancers, and the BCL2-selective inhibitor ABT-199 (venetoclax) has obtained clinical success. However, MCL1 can promote drug resistance and overall cancer cell survival. Thus, there is a critical need to develop an effective drug that antagonizes MCL1. However, most putative MCL1 inhibitors have been misclassified as they fail to directly inhibit MCL1 in cells, but rather induce the pro-apoptotic protein NOXA. We have investigated three putative MCL1 inhibitors: MIM1, UMI-77, and A-1210477. All three compounds were developed in cell-free assays and then found to be cytotoxic, and hence assumed to directly target MCL1 in cells. Here, we investigated whether these compounds directly inhibit MCL1 or inhibit MCL1 indirectly through the induction of NOXA. Both MIM1- and UMI-77-induced NOXA through the unfolded protein response pathway, and sensitized leukemia cells to ABT-199; this cytotoxicity was dependent on NOXA suggesting that these compounds do not directly target MCL1. A-1210477 was the only compound that did not induce NOXA, but it still sensitized cells to ABT-199. A-1210477 induced accumulation of MCL1 protein consistent with it binding and preventing MCL1 degradation. However, at concentrations used in several prior studies, A-1210477 also induced cytochrome c release, caspase activation, and apoptosis in a BAX/BAK-independent manner. Furthermore, the release of cytochrome c occurred without loss of mitochondrial membrane potential. This apoptosis was extremely rapid, sometimes occurring within 0.5-1 h. Hence, we have identified a novel mechanism of apoptosis that circumvents the known mechanisms of cytochrome c release. It remains to be determined whether these unexpected mechanisms of action of putative BH3 mimetics will have therapeutic potential.
Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Biomimética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Citocromos c/metabolismo , Humanos , Indóis/farmacologia , Células Jurkat , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Tioglicolatos/farmacologia , Células U937 , Regulação para Cima/genéticaRESUMO
Rac GTPases have oncogenic roles in cell growth, survival, and migration. We tested response to the Rac inhibitor EHT1864 in a panel of breast cancer cell lines. EHT1864-induced growth inhibition was associated with dual inhibition of the PI3K/AKT/mTORC1 and MEK/ERK pathways. Breast cancer cells harboring PIK3CA mutations or HER2 overexpression were most sensitive to Rac inhibition, suggesting that such oncogenic alterations link Rac activation with PI3K/AKT/mTORC1 and MEK/ERK signaling. Interestingly, EHT1864 decreased activation of the mTORC1 substrate p70S6K earlier than AKT inhibition, suggesting that Rac may activate mTORC1/p70S6K independently of AKT. Comparison of the growth-inhibitory profile of EHT1864 to 137 other anti-cancer drugs across 656 cancer cell lines revealed significant correlation with the p70S6K inhibitor PF-4708671. We confirmed that Rac complexes contain MEK1/2 and ERK1/2, but also contain p70S6K; these interactions were disrupted by EHT1864. Pharmacokinetic profiles revealed that EHT1864 was present in mouse plasma at concentrations effective in vitro for approximately 1 h after intraperitoneal administration. EHT1864 suppressed growth of HER2+ tumors, and enhanced response to anti-estrogen treatment in ER+ tumors. Further therapeutic development of Rac inhibitors for HER2+ and PIK3CA-mutant cancers is warranted.
Assuntos
Neoplasias da Mama/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Humanos , MAP Quinase Quinase Quinases/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Microtubule targeting agents, such as vinblastine, are usually thought to arrest cells in mitosis and subsequently induce apoptosis. However, they can also cause rapid induction of apoptosis in a cell-cycle phase independent manner. BNC105 is a novel vascular and microtubule disrupting drug that also induces apoptosis rapidly but with markedly increased potency compared to vinca alkaloids and combretastatin A4. BNC105 binds to the colchicine-binding site on tubulin resulting in activation of c-Jun N-terminal kinase (JNK), phosphorylation of ATF2, and induction of ATF3 and Noxa leading to acute apoptosis in chronic lymphocytic leukemia (CLL) cells. Apoptosis induced by BNC105 is dependent upon both JNK activation and Noxa induction. Normal leukocytes and one CLL sample also exhibited JNK activation but not Noxa induction and were resistant to BNC105. This study emphasizes the importance of Noxa and JNK for induction of apoptosis in CLL cells by microtubule targeting drugs, and highlights the potential of BNC105 as a potent therapeutic to treat haematopoietic malignancies.
Assuntos
Anisóis/farmacologia , Apoptose/efeitos dos fármacos , Benzofuranos/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Microtúbulos/efeitos dos fármacos , Moduladores de Tubulina/farmacologia , Linhagem Celular Tumoral , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Sistema de Sinalização das MAP Quinases , Microtúbulos/metabolismo , Estilbenos/farmacologia , Transfecção , Vimblastina/farmacologiaRESUMO
PURPOSE: Phosphatidylinositol 3-kinase (PI3K) inhibitors are being developed for the treatment of estrogen receptor α (ER)-positive breast cancer in combination with antiestrogens. Understanding the temporal response and pharmacodynamic effects of PI3K inhibition in ER(+) breast cancer will provide a rationale for treatment scheduling to maximize therapeutic index. EXPERIMENTAL DESIGN: Antiestrogen-sensitive and antiestrogen-resistant ER(+) human breast cancer cell lines and mice bearing PIK3CA-mutant xenografts were treated with the antiestrogen fulvestrant, the PI3K inhibitor GDC-0941 (pictilisib; varied doses/schedules that provided similar amounts of drug each week), or combinations. Cell viability, signaling pathway inhibition, proliferation, apoptosis, tumor volume, and GDC-0941 concentrations in plasma and tumors were temporally measured. RESULTS: Treatment with the combination of fulvestrant and GDC-0941, regardless of dose/schedule, was significantly more effective than that with single-agent treatments in fulvestrant-resistant tumors. Short-term, complete PI3K inhibition blocked cell growth in vitro more effectively than chronic, incomplete inhibition. Longer-term PI3K inhibition hypersensitized cells to growth factor signaling upon drug withdrawal. Different schedules of GDC-0941 elicited similar tumor responses. While weekly high-dose GDC-0941 with fulvestrant continuously suppressed PI3K signaling for 72 hours, inducing a bolus of apoptosis and inhibiting proliferation, PI3K reactivation upon GDC-0941 washout induced a proliferative burst. Fulvestrant with daily low-dose GDC-0941 metronomically suppressed PI3K for 6 to 9 hours/day, repeatedly inducing small amounts of apoptosis and temporarily inhibiting proliferation, followed by proliferative rebound compared with fulvestrant alone. CONCLUSIONS: Continuous and metronomic PI3K inhibition elicits robust anticancer effects in ER(+), PIK3CA-mutant breast cancer. Clinical exploration of alternate treatment schedules of PI3K inhibitors with antiestrogens is warranted. Clin Cancer Res; 22(9); 2250-60. ©2016 AACRSee related commentary by Toska and Baselga, p. 2099.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/genética , Receptor alfa de Estrogênio/genética , Mutação/genética , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Fulvestranto , Humanos , Indazóis/farmacologia , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Mutação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Índice TerapêuticoRESUMO
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.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Compostos de Bifenilo/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/genética , Nitrofenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/farmacologia , Vimblastina/farmacologia , Antineoplásicos Fitogênicos/toxicidade , Apoptose/efeitos dos fármacos , Apoptose/genética , Compostos de Bifenilo/toxicidade , Plaquetas/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfócitos/efeitos dos fármacos , Nitrofenóis/toxicidade , Piperazinas/farmacologia , Piperazinas/toxicidade , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células Estromais/metabolismo , Sulfonamidas/toxicidade , Moduladores de Tubulina/farmacologia , Moduladores de Tubulina/toxicidade , Vimblastina/toxicidadeRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Flavonoides/farmacologia , Piperidinas/farmacologia , Compostos de Piridínio/farmacologia , Vimblastina/farmacologia , Antracenos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Óxidos N-Cíclicos , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Células HL-60 , Humanos , Immunoblotting , Indolizinas , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células Jurkat , Proteína de Sequência 1 de Leucemia de Células Mieloides , Paclitaxel/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células U937 , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Chemotherapeutic agents modify intracellular signaling that culminates in the inhibition of Bcl-2 family members and initiates apoptosis. Inhibition of the extracellular signal-regulated kinase by PD98059 dramatically accelerates vinblastine-mediated apoptosis in ML-1 leukemia with cells dying in 4 hours from all phases of the cell cycle. Inhibition of protein synthesis by cycloheximide also markedly accelerated vinblastine-induced apoptosis, showing that the proteins required for this acute apoptosis are constitutively expressed. Vinblastine induced the rapid induction of Mcl-1 that was inhibited by PD98059 and cycloheximide. No change in Bcl-2 or Bcl-X was observed. We hypothesize that ML-1 cells use Mcl-1 for protection from the rapid vinblastine-induced apoptosis. This was confirmed by targeting Mcl-1 with short hairpin RNA. We also investigated the response of 13 other leukemia and lymphoma cell lines and cells from seven chronic lymphocytic leukemia patients. Four cell lines and all chronic lymphocytic leukemia cells were killed in 6 hours by vinblastine alone. Two additional cell lines were sensitized to vinblastine by PD98059, which suppressed Mcl-1. This acute apoptosis either alone or in combination with PD98059 required vinblastine-mediated activation of c-Jun-NH(2)-terminal kinase. PD98059 did not suppress Mcl-1 in other cell lines whereas sorafenib did, but this did not sensitize the cells to vinblastine, suggesting that the acute apoptosis varies depending on which Bcl-2 protein mediates protection. Most of the cell lines were sensitized to vinblastine by cycloheximide, suggesting that inhibition of a short-lived protein in addition to Mcl-1 can acutely sensitize cells. These results suggest several clinical strategies that might provide an effective therapy for selected patients. Mol Cancer Ther; 9(4); 791-802. (c)2010 AACR.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Leucemia/patologia , Linfoma/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Vimblastina/farmacologia , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Citoproteção/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Flavonoides/farmacologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Leucemia/enzimologia , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células MieloidesRESUMO
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).