RESUMO
Although migrations are essential for soil microorganisms to exploit scarce and heterogeneously distributed resources, bacterial mobility in soil remains poorly studied due to experimental limitations. In this study, time-lapse images collected using live microscopy techniques captured collective and coordinated groups of B. subtilis cells exhibiting "crowd movement". Groups of B. subtilis cells moved through transparent soil (nafion polymer with particle size resembling sand) toward plant roots and re-arranged dynamically around root tips in the form of elongating and retracting "flocks" resembling collective behaviour usually associated with higher organisms (e.g., bird flocks or fish schools). Genetic analysis reveals B. subtilis flocks are likely driven by the diffusion of extracellular signalling molecules (e.g., chemotaxis, quorum sensing) and may be impacted by the physical obstacles and hydrodynamics encountered in the soil like environment. Our findings advance understanding of bacterial migration through soil matrices and expand known behaviours for coordinated bacterial movement.
Assuntos
Areia , Solo , Bactérias/genética , Polímeros , Percepção de QuorumRESUMO
The tumour microenvironment is believed to be involved in development, growth, metastasis, and therapy resistance of many cancers. Here we show survivin, a member of the inhibitor of apoptosis protein (IAP) family, implicated in apoptosis inhibition and the regulation of mitosis in cancer cells, exists in a novel extracellular pool in tumour cells. Furthermore, we have constructed stable cell lines that provide the extracellular pool with either wild-type survivin (Surv-WT) or the previously described dominant-negative mutant survivin (Surv-T34A), which has proven pro-apoptotic effects in cancer cells but not in normal proliferating cells. Cancer cells grown in conditioned medium (CM) taken from Surv-WT cells absorbed survivin and experienced enhanced protection against genotoxic stresses. These cells also exhibited an increased replicative and metastatic potential, suggesting that survivin in the tumour microenvironment may be directly associated with malignant progression, further supporting survivin's function in tumourigenesis. Alternatively, cancer cells grown in CM taken from the Surv-T34A cells began to apoptose through a caspase-2- and caspase-9-dependent pathway that was further enhanced by the addition of other chemo- and radiotherapeutic modalities. Together our findings suggest a novel microenvironmental function for survivin in the control of cancer aggressiveness and spread, and should result in the genesis of additional cancer treatment modalities.
Assuntos
Apoptose , Proteínas Associadas aos Microtúbulos/fisiologia , Metástase Neoplásica , Neoplasias/patologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Proteínas Inibidoras de Apoptose , Potencial da Membrana Mitocondrial , Proteínas Associadas aos Microtúbulos/análise , Invasividade Neoplásica , SurvivinaRESUMO
Insulin-like growth factor II (IGF-II) plays a pivotal role in fetal and cancer development by signaling through the IGF-I and insulin receptors and activating the estrogen signaling cascade. We previously showed that precursor IGF-II (proIGF-II, the predominant form expressed in cancer) and not mature IGF-II (mIGF-II) blocks resveratrol (RSV) (a phytoalexin/anticancer agent)-induced cell death in MCF-7 cells. We hypothesize that proIGF-II regulates antiapoptotic proteins and/or the mitochondria to inhibit RSV actions and promote cell survival. This study examines the effect of mIGF-II and proIGF-II on survivin expression and mitochondrial polarization in response to RSV. RSV inhibits survivin expression and stimulates mitochondrial depolarization, caspase 7 activation and cell death. These effects were completely blocked by the addition of proIGF-II. RSV treatment had no effect on transfected MCF-7 cells constitutively expressing proIGF-II, while IGF-II siRNA transfection decreased survivin levels. Our results provide new insights for the potential use of proIGF-II as target for new anticancer therapies.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Fator de Crescimento Insulin-Like II/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Precursores de Proteínas/fisiologia , Estilbenos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Humanos , Proteínas Inibidoras de Apoptose , Potencial da Membrana Mitocondrial , Mitocôndrias/fisiologia , Processamento de Proteína Pós-Traducional , Resveratrol , Transdução de Sinais/efeitos dos fármacos , SurvivinaRESUMO
Bryostatin 1 (bryo 1) is known to induce the differentiation and cell cycle arrest of human lymphoid leukemia cells in vitro. The extracellular signal-regulated kinase (ERK), originally identified as a participant in mitogenic signaling, has recently been implicated in the signaling of cellular differentiation. To examine the role of the ERK/mitogen-activated protein (MAP) kinase pathway in B-lymphoid cell differentiation of the Reh Acute Lymphoblastic Leukemia cell line, the effects of bryo 1 on ERK activation were determined. On bryo 1 treatment, the activity of ERK2 (p42) rapidly increased, with ERK1 (p44) protein levels remaining constant. p44/42 immunoprecipitates from lysates of bryo 1-treated cells had increased their ability to phosphorylate the transcription factor Elk-1. Constitutive AP-1 activity was shown to be potentiated after bryo 1 treatment using electrophoretic mobility shift assays. The protein composition of the AP-1 transcription factor complex activated by bryo 1 was analyzed using supershift analysis with specific antibodies against c-Fos, Fos B, c-Jun, Jun B, and Jun D proteins. Supershift analysis revealed that the bryo 1-induced AP-1 complex was composed predominantly of Fos B and Jun D. Therefore, we evaluated the effects of inhibiting MAP/ERK kinase (MEK) on both DNA binding and cellular differentiation. Treatment of Reh cells with 20 microM PD98059, a specific inhibitor of MEK, inhibited bryo 1-induced ERK activity and DNA binding. Furthermore, PD98059 blocked the bryo 1-induced differentiation of Reh cells, as assessed by a number of features associated with lymphoid differentiation, including changes in morphology, cell growth arrest, attachment, and increased expression of the leukocyte integrin CD11c. Moreover, transient transfection of Reh cells with antisense MAP kinase oligonucleotides blocked bryo 1-induced expression of CD11c. Our analysis also shows that CD11c's gene promoter activity is augmented by bryo 1. Therefore, we conclude that activation of the MEK/ERK signaling pathway is necessary for bryo 1-induced differentiation of the pre-B Acute Lymphoblastic Leukemia cell line Reh.
Assuntos
Lactonas/metabolismo , Sistema de Sinalização das MAP Quinases , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-fos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Western Blotting , Briostatinas , Diferenciação Celular , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Citometria de Fluxo , Humanos , Integrina alfaXbeta2/biossíntese , Luciferases/metabolismo , Macrolídeos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
We have constructed a replication-deficient adenovirus encoding a nonphosphorylatable Thr(34)-->Ala mutant of the apoptosis inhibitor survivin (pAd-T34A) to target tumor cell viability in vitro and in vivo. Infection with pAd-T34A caused spontaneous apoptosis in cell lines of breast, cervical, prostate, lung, and colorectal cancer. In contrast, pAd-T34A did not affect cell viability of proliferating normal human cells, including fibroblasts, endothelium, or smooth muscle cells. Infection of tumor cells with pAd-T34A resulted in cytochrome c release from mitochondria, cleavage of approximately 46-kDa upstream caspase-9, processing of caspase-3 to the active subunits of approximately 17 and 19 kDa, and increased caspase-3 catalytic activity. When compared with chemotherapeutic regimens, pAd-T34A was as effective as taxol and considerably more effective than adriamycin in induction of tumor cell apoptosis and enhanced taxol-induced cell death. In three xenograft breast cancer models in immunodeficient mice, pAd-T34A suppressed de novo tumor formation, inhibited by approximately 40% the growth of established tumors, and reduced intraperitoneal tumor dissemination. Tumors injected with pAd-T34A exhibited loss of proliferating cells and massive apoptosis by in situ internucleosomal DNA fragmentation. These data suggest that adenoviral targeting of the survivin pathway may provide a novel approach for selective cancer gene therapy.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Inibidores de Cisteína Proteinase , Proteínas Associadas aos Microtúbulos , Neoplasias/terapia , Adenovírus Humanos , Animais , Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Ciclo Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/uso terapêutico , Doxorrubicina/farmacologia , Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose , Camundongos , Camundongos SCID , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias , Neoplasias Experimentais , Paclitaxel/farmacologia , Survivina , Células Tumorais CultivadasRESUMO
Combretastatin A-4 prodrug (CA4P) is a new antitubulin agent currently in phase I/II clinical trials against solid tumors. We have previously reported on the in vitro activity of CA4P against a panel of malignant human B-lymphoid cell lines. In this study, we investigated the antitumor and the antiangiogenic activity of CA4P in our diffuse large cell lymphoma WSU-DLCL2-SCID mouse model. WSU-DLCL2 cells (10(7)) were injected s.c. into 5-week-old female ICR-SCID mice. Tumor-bearing mice were treated at the CA4P maximum tolerated dose (MTD) of 800 mg/kg in different dose/schedules. CA4P showed significant antitumor activity against this lymphoma model. Best results were seen when MTD was given in two and four divided doses (400 and 200 mg/kg, respectively). CA4P given in four divided doses (4 x 200 mg/kg) showed a log10 kill of 1.01, T/C of 11.7% and T-C of 12 days. Immunohistochemical staining using anti-CD31 antibody after 6, 24, 48 and 120 h treatment revealed a significant decrease in the number of tumor blood vessels after 24 h (about 80%). Only the periphery of treated tumors revealed the presence of blood vessels. Morphological examination of the tumors after tetrachrome staining showed a necrotic center in tumors of CA4P-treated animals. New blood vessel formation was noted to emerge in tumor tissues as early as 48 h following a single dose of CA4P. The G2/M arrest observed in vitro was not detected in vivo indicating predominance of the antiangiogenic effects with regard to antitumor efficacy in vivo. We conclude that CA4P has antiangiogenic activity in this lymphoma model and the use of this agent should be explored clinically in the treatment of non-Hodgkin's lymphoma.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Linfoma não Hodgkin/tratamento farmacológico , Pró-Fármacos/farmacologia , Estilbenos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Feminino , Humanos , Linfoma não Hodgkin/patologia , Dose Máxima Tolerável , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Neovascularização Patológica/tratamento farmacológico , Pró-Fármacos/administração & dosagem , Estilbenos/administração & dosagem , Taxa de SobrevidaRESUMO
Bryostatin 1 (bryo 1) has been shown to potentiate the anti-tumor activity of 2-chloro-2-deoxyadenosine (2-CdA) in chronic lymphocytic leukemia (CLL) and in the WSU-CLL cell line. However, like resistant CLL, WSU-CLL cells lose their sensitivity to bryo 1/2-CdA treatment. We report that 2-CdA-induced IAP expression may be a possible mechanism whereby resistance to apoptosis is acquired in these cells. In WSU-CLL cells, three members of the Inhibitors of Apoptosis (IAP) family were identified. Bryo 1 treatment of WSU-CLL cells leads to initiation of the apoptotic cascade and induced a marginal increase in XIAP protein expression. In contrast, 2-CdA treatment, alone or in combination with bryo 1, induced a substantial increase in survivin and XIAP proteins and phosphorylation of BAD. Bryo 1 alone induced caspase-7 and -9 dependent [poly ADP-ribose] polymerase (PARP) cleavage, while sequential treatment with bryo 1 (72 h) followed by 2-CdA (24 h) induced caspase-3,-7, and -9 dependent PARP cleavage and increased apoptosis. Although exposure to bryo 1 initiated apoptotic events, apoptosis was first enhanced by 2-CdA, and then reversed in a time-dependent manner by 2-CdA-induced expression of survival proteins. Taken together, resistance to bryo 1/2-CdA treatment may be the result of 2-CdA-induced IAP inhibition of the intrinsic apoptotic pathway caspases.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Lactonas/farmacologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , 2-Cloroadenosina/análogos & derivados , 2-Cloroadenosina/farmacologia , Idoso , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/fisiologia , Briostatinas , Desoxiadenosinas/farmacologia , Ativadores de Enzimas/farmacologia , Humanos , Macrolídeos , Masculino , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
Combretastatin A-4 (CA-4) is one of a family of compounds isolated from the South African willow tree Combretum caffrum. CA-4 was found to be active against murine melanoma and a variety of other human solid tumors. For the first time, we report the effect of CA-4 against a panel of malignant human B-lymphoid cell lines [early pre-B acute lymphoblastic leukemia (Reh), diffuse large cell lymphoma (WSU-DLCL2), chronic lymphocytic leukemia (WSU-CLL) and Waldenstrom's macroglobulinemia (WSU-WM)]. Our results indicate, using the prodrug form of CA-4, a concentration-dependent growth inhibition in all tested cell lines, although WSU-DLCL2 was more sensitive. Exposure to 4 nM CA-4 for 96 h induced 77% growth inhibition in Reh, 86% in WSU-CLL and 92% in WSU-WM. When used against the WSU-DLCL2 cell line, this same concentration of CA-4 was completely toxic. Morphological examination showed CA-4 induced the formation of giant, multinucleated cells, a phenomenon commonly found in mitotic catastrophe. Only minimal numbers of cells showing characteristics of apoptosis were detected. In WSU-DLCL2 cells, CA-4 (3 nM) induced the highest apoptosis (5%) after 48 h, while the percentage of dead cells was approximately 47%. Exposure of Reh, WSU-CLL, WSU-WM and WSU-DLCL2 cells for 24 h to 5 nM CA-4 induced 19, 28, 57 and 75% G2/M arrest, as determined by flow cytometry, respectively. Based on these preliminary studies, we believe that mitotic catastrophe is the predominant mechanism by which CA-4 induces cell death rather than apoptosis. Further studies to elucidate the mechanisms of CA-4 activity in vitro and in vivo are currently under investigation in our laboratory.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Linfoma de Células B/tratamento farmacológico , Pró-Fármacos/farmacologia , Estilbenos/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Citometria de Fluxo , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Mitose/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
WSU-CLL cells, a fludarabine resistant B-cell chronic lymphocytic leukemia cell line, has been shown to exhibit enhanced sensitivity to 2-chlorodeoxyadenosine (2-CdA) following 48-72 h exposure to bryostatin 1. For 2-CdA to manifest its chemotherapeutic activity, it must first enter the cell through one of several specific nucleoside transporter systems. We present data to show that bryostatin 1-induced enhanced influx of 2-CdA is in part the result of bryostatin 1-induced modulation of nucleoside transporters in WSU-CLL cells. The bi-directional equilibrative NBMPR sensitive transporters in WSU-CLL cells were significantly down-regulated 90 min post-exposure to 1-200 nM bryostatin 1. This down-regulation was evident up to 144 h. In contrast, WSU-CLL cells exhibited a transient increase in Na+-dependent concentrative 2-CdA influx from 48 to 96 h after bryostatin 1 exposure which was evident for a longer duration than that accounted for by the increase in deocycytidine kinase activity. These data may, in part, explain the enhanced efficacy of 2-CdA seen in WSU-CLL cells following 48-72 h exposure to bryostatin 1. It may raise questions as to the importance of the bi-directional transporters in determining the resistance or sensitivity of CLL cells to 2-CdA or other nucleoside analogues.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Transporte/metabolismo , Cladribina/metabolismo , Lactonas/farmacologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas de Membrana/metabolismo , Idoso , Transporte Biológico Ativo/efeitos dos fármacos , Briostatinas , Desoxicitidina Quinase/metabolismo , Dipiridamol/farmacologia , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Macrolídeos , Masculino , Proteínas de Transporte de Nucleosídeos , Fosforilação , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Células Tumorais Cultivadas , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
The ubiquitin-mediated proteolytic system has been implicated in the turnover of a number of intracellular proteins. In the present study, we investigated the novelty and potential role of bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan, Bugula neritina, in inducing the ubiquitin-mediated proteolysis of the oncoprotein Bcl-2. Immunoprecipitation and immunoblotting analyses revealed that Bcl-2 is ubiquitinated following exposure of the acute lymphoblastic leukemia (ALL) cell line Reh to 1 nM bryostatin 1. Bcl-2 protein rapidly decreases to 50% of that recorded in the control after 24 h of bryostatin 1 treatment. In the subsequent 24 h, Bcl-2 protein again rapidly decreases to 6% of its pre-bryostatin 1 level at which time a plateau is reached and maintained for another 72 h. Furthermore, ubiquitin-Bcl-2 conjugates are detected in untreated as well as bryostatin 1 treated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of Bcl-2. However, ubiquitin-Bcl-2 conjugates increase in a time-dependent manner following bryostatin 1 treatment. Lactacystin, which inhibits the proteinase activities of the proteasome, inhibited the bryostatin 1-induced decrease of Bcl-2 protein. The effect of bryostatin 1 on the proteolytic efficiency of the 26S proteasome in Reh cell extracts was also investigated and shown to increase following 1 h of bryostatin 1 treatment. Proteolytic activity reached its highest point by 3 h, and subsequently returned to control levels by 12 h, post-bryostatin 1 treatment. In addition, bryostatin 1 treatment of the Reh cell line decreased expression of bcl-2 mRNA within 3 h. However, bcl-2 mRNA expression returned after 24 h. We speculate that this decrease in mRNA together with increased 26S proteolytic activity accounts for the initial rapid decrease recorded in Bcl-2 protein. These findings indicate that bryostatin 1 treatment of Reh ALL cells decreases Bcl-2 expression through two processes: a) enhanced Bcl-2 protein degradation through the activation of the ubiquitin-proteasome pathway and b) decreased bcl-2 mRNA expression.
Assuntos
Antineoplásicos/farmacologia , Cisteína Endopeptidases/metabolismo , Lactonas/farmacologia , Complexos Multienzimáticos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ubiquitinas/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Northern Blotting , Western Blotting , Briostatinas , Extratos Celulares , Humanos , Macrolídeos , Testes de Precipitina , Leucemia-Linfoma Linfoblástico de Células Precursoras , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Células Tumorais CultivadasRESUMO
The incidence of non-Hodgkin's lymphoma has been increasing at a rate of 4% per year since 1950; more than 62,000 cases will be diagnosed in the United States in 2000. Diffuse large cell lymphoma (DLCL) is the prototype of curable non-Hodgkin's lymphoma. Empirically designed chemotherapy regimens did not increase the cure rate of 30-40% achieved by the original four-drug regimen introduced in the 1970s [cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)]. We studied the antitumor effects of the CHOP regimen alone or in combination with a unique protein kinase C activator, bryostatin 1, on a xenograft model for resistant DLCL in mice with severe combined immune deficiency (WSU-DLCL2-SCID). In this model, the efficacy of bryostatin 1 given at 75 microg/kg, i.p., alone for 1 or 2 days [B(1x) and B(2x)]was compared with the efficacy of CHOP alone, bryostatin 1 + CHOP (B+CHOP) given concurrently, bryostatin 1 for 1 day followed by CHOP on day 2 [B(1x)-CHOP], and bryostatin 1 for 2 days followed by CHOP on day 3 [B(2x)-CHOP]. CHOP doses were as follows: (a) cyclophosphamide, 40 mg/kg, i.v.; (b) doxorubicin, 3.3 mg/kg, i.v.; (c) vincristine, 0.5 mg/kg, i.v.; and (d) prednisone, 0.2 mg/kg, every day for 5 days, p.o. Tumor growth inhibition (T/C), tumor growth delay (T-C), and log10 kill for B(1x), B(2x), CHOP, B+CHOP, B(1x)-CHOP and B(2x)-CHOP were 49%, 39%, 25.8%, 15.1%, 14.6%, and 12%; 6, 7, 16, 25, 12, and 15 days; and 0.6, 0.5, 2.2, 3.6, 1.7, and 2.0, respectively. To begin elucidating the mechanism whereby bryostatin 1 potentiated the effects of CHOP in the mouse model; we studied the effect of bryostatin 1 on Bax, Bcl-2, and poly(ADP-ribose) polymerase proteins in vitro and in vivo. Bax protein increased in a time-dependent manner without any measurable change in Bcl-2 expression. However, significant cleavage of the preapoptotic marker poly(ADP-ribose) polymerase was not recorded, and the percentage of apoptotic cells detected by flow cytometry increased only slightly (approximately 8%) after 96 h of bryostatin 1 exposure. The in vitro and in vivo results emphasize the superiority of combining bryostatin 1 with the CHOP regimen against the WSU-DLCL2 model. One possible mechanism may be the modulatory effects of bryostatin 1 on the Bax:Bcl-2 family of apoptosis-regulatory proteins. The use of this combination should be further explored clinically in the treatment of lymphoma.
Assuntos
Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Lactonas/administração & dosagem , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Prednisolona/administração & dosagem , Vincristina/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Briostatinas , Ativação Enzimática , Citometria de Fluxo , Humanos , Macrolídeos , Camundongos , Camundongos SCID , Transplante de Neoplasias , Poli(ADP-Ribose) Polimerases/biossíntese , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Fatores de Tempo , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
Previous studies have shown that bryostatin 1 induces a decrease in the expression of the antiapoptotic protooncogene Bcl-2 in the human acute lymphoblastic leukemia (ALL) cell line Reh. This down-regulation has been shown to reduce drug resistance of the Reh cells to anti-tubulin polymerization agents. In the present study we investigated the effect of bryostatin 1 alone and in combination with novel anti-tubulin agents (dolastatin 10 and auristatin PE) and the chemotherapeutic vincristine on the inhibitor of apoptosis protein cIAP-1. Cells were cultured with bryostatin 1 (1 nM), dolastatin 10 (0.1 ng/ml), auristatin PE (0.1 ng/ml), or vincristine (0.5 ng/ml) alone or the combination of these anti-tubulins with bryostatin 1. Western blots were conducted to assess the effects of the above agents on cIAP-1 protein level. Flow-cytometric analysis [7-amino-actinomycin D (7AAD)] was conducted to assess apoptosis as well as staining for morphology using tetrachrome stain. Our results show that cIAP-1 is induced in a time-dependent fashion after bryostatin 1 exposure up to 72 h. However, upon treatment of cells with a combination of bryostatin 1 and dolastatin 10 or auristatin PE, the induction of cIAP-1 was abolished, leading to a significant increase in apoptosis. The initial 24- and 48-h reduction in cIAP-1 protein level recorded in the bryostatin 1 and vincristine combination recovered to control levels by 72 h. We believe that this phenomenon is responsible for the reduced apoptosis recorded in this combination. Results of this study should prove useful in guiding the clinical application of these novel agents in the treatment of ALL.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Lactonas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas/metabolismo , Briostatinas , Depsipeptídeos , Regulação para Baixo/efeitos dos fármacos , Citometria de Fluxo , Humanos , Proteínas Inibidoras de Apoptose , Macrolídeos , Oligopeptídeos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células Tumorais Cultivadas , Vincristina/farmacologiaRESUMO
The ratio of Bax to Bcl-2 protein can determine whether cells will die via apoptosis or be protected from it. Reh was found to express a high basal level of Bcl-2 but was lacking of Bax protein expression. Treatment with bryostatin 1 induced a down-regulation in Bcl-2 protein that was not accompanied by an obvious Bax protein induction or apoptosis. These results suggest that a decreased level of Bcl-2 alone in this cell line is not sufficient for apoptosis induction. In an effort to identify the mechanism whereby apoptosis could be induced in this ALL model, we treated Reh cells with three microtubule inhibitors: dolastatin 10, auristatin PE and vincristine, in the presence and absence of bryostatin 1. When used alone, only dolastatin 10 induced apoptosis that was detected morphologically, and by flow cytometry. Western blots revealed that dolastatin 10-induced apoptosis was accompanied by the induction of Bax protein and the reduction in Bcl-2 protein. Auristatin PE and vincristine induced both Bax and Bcl-2 protein, leaving the Bax:Bcl-2 ratio constant. Reh cells pretreated for 24 h with bryostatin 1 followed by dolastatin 10, auristatin PE or vincristine showed significant apoptosis which was accompanied by Bcl-2 protein down regulation and Bax protein up regulation. We conclude that: (1) expression of bax is necessary for apoptosis-induction in this model; (2) a decrease in Bcl-2 level alone is not sufficient and might not be necessary for apoptosis-induction; and (3) the ratio of Bax:Bcl-2 plays a critical role in susceptibility to apoptosis in Reh cells. The results from this study should prove useful in guiding the clinical application of these novel agents in the treatment of acute lymphoblastic leukemia.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Lactonas/farmacologia , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Vincristina/farmacologia , Antineoplásicos/uso terapêutico , Briostatinas , Linfoma de Burkitt/tratamento farmacológico , Humanos , Lactonas/uso terapêutico , Macrolídeos , Oligopeptídeos/uso terapêutico , Células Tumorais Cultivadas , Vincristina/uso terapêutico , Proteína X Associada a bcl-2RESUMO
Waldenstrom's macroglobulinemia (WM) is an uncommon lymphoproliferative disease which remains incurable with current treatment protocols. We have previously established a permanent WM cell line, WSU-WM, which grows as a xenograft in severe combined immune deficient (SCID) mice. In this study, we investigated the anti-tumor effects of auristatin PE (a structural modification of the marine, shell-less mollusk peptide constituent dolastatin 10). WSU-WM cells were cultured in RPMI-1640 at a concentration of 2x10(5) cells/ml using 24-well plates. Auristatin PE or dolastatin 10 were added to triplicate wells and cell count and viability were assessed after 24, 48 and 72 h. Results showed that both agents were active against WSU-WM, and were able to induce complete growth inhibition at 100 pg/ml. The efficacy of these agents in vivo was evaluated using the WSU-WM SCID mouse xenograft model. Auristatin PE and dolastatin 10 were given i.v. via tail vein at 2.0 mg/kg and 0.2 mg/kg, respectively. The agents were given every second day for three injections which represent the maximum tolerated doses. Tumor growth inhibition (T/C), tumor growth delay (T-C), and log10 kill for auristatin PE and dolastatin 10 were 0%, 18 days, 2.83 and 67%, 2 days, 0.06, respectively. Based on these animal results, dolastatin 10 was inactive while auristatin PE was highly active. We therefore focused further investigation on auristatin PE to understand some of its mechanisms of action. Using two flow cytometry assays, propidium iodide for cell cycle analysis and 7-amino actinomycin D (7AAD) to detect apoptosis, we were able to demonstrate that auristatin PE at 10 pg/ml after 24 h arrested 50% of WSU-MW cells in G2M. Concomitantly, 31% of auristatin PE-treated cells entered apoptosis. By 72 h, greater than 75% of the cells became apoptotic. The activity of auristatin PE should be evaluated in other tumor types and in clinical trials.
Assuntos
Antineoplásicos/uso terapêutico , Oligopeptídeos/uso terapêutico , Indução de Remissão/métodos , Tubulina (Proteína)/efeitos dos fármacos , Macroglobulinemia de Waldenstrom/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Biopolímeros , Divisão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Mitose/efeitos dos fármacos , Imunodeficiência Combinada Severa , Transplante HeterólogoRESUMO
WSU-CLL is a de novo fludarabine resistant cell line established from a patient with advanced chronic lymphocytic leukemia (CLL) refractory to chemotherapy including fludarabine (Flud). Our previous studies indicate that bryostatin 1 (Bryo 1) induces differentiation of WSU-CLL and increases the ratio of dCK/5'-NT activity and Bax/Bcl-2. This study tests the hypothesis that Bryo 1-differentiated cells are more susceptible to Flud than the parent WSU-CLL cells. Flud, given sequentially after Bryo 1, in vitro and in vivo animal studies resulted in significantly higher rates of growth inhibition and improved animal survival. Flud at 100 to 600 nM exhibited a dose-dependent growth inhibitory effect on the WSU-CLL cell line. The sequential exposure to Bryo 1 (10 nM for 72 h) followed by Flud (100 nM) resulted in significantly higher rates of growth inhibition than either the reverse addition of these two agents or each agent alone, but was not significantly different than the concurrent addition of Bryo 1 + Flud. Using 7-amino-actinomycin D staining and flow cytometry, apoptosis was seen in 40.8% of cells treated with Bryo 1 (10 nM, 72 h) followed by Flud, compared with Flud (100 nM, 72 h) followed by Bryo 1 (18.1%). To demonstrate that Bryo 1 enhancement of Flud efficacy was not restricted to in vitro culture, we used the WSU-CLL xenograft model in mice with severe combined immune deficiency (SCID). Bryo 1 + Flud at the maximum tolerated doses (75 microg/kg i.p. and 200 mg/kg i.v., respectively) were administered to mice in different combinations. The survival in days, the tumor growth inhibition ratio (T/C), the tumor growth delay (T-C) in days, log10 kill, as well as mean tumor weight (mtw) of mice treated with Bryo 1 followed by Flud, were significantly better than control and other groups. T/C%, T-C, log10 kill and mtw were as follows: Bryo 1 (36.8%, 10 days, 0.8, 375 mg); Flud (100%, 0. 0 day, 0.0, 1130 mg); Bryo 1 + Flud (14.3%, 12 days, 0.95, 288 mg); Bryo 1 followed by Flud (4.6%, 17 days, 1.35, 35 mg); Flud followed by Bryo (40.3%, 10 days, 0.80, 175 mg). We conclude that: i) Bryo 1 sensitizes WSU-CLL cells to Flud and enhances apoptosis; ii) the sequential treatment with Bryo 1 followed by Flud resulted in higher anti-tumor activity compared with either agent alone, in combination, or the reverse addition of these agents and iii) these results are comparable to those of Bryo 1 followed by 2-CdA suggesting common pathway(s) of interaction between Bryo 1 and purine analogues.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Animais , Apoptose , Briostatinas , Divisão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Humanos , Lactonas/administração & dosagem , Macrolídeos , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Transplante de Neoplasias , Transplante Heterólogo , Células Tumorais Cultivadas , Vidarabina/administração & dosagem , Vidarabina/análogos & derivadosRESUMO
The protein metabolism and [3H]-uridine uptake of thyroid and adenohypophysis and the kinetics of pituitary TSH rebound (PTR) were studied in goitrous female rats (fed propylthiouracil, PTU: for 7-12 weeks) following single, iv injections of L-thyroxine (T4: 0.8 to 200 mug). Goitrogenesis was associated with reduced protein concentration and enhanced uptake of [3H] uridine in both glands. Plasma levels of TSH were invariably elevated but stores in the adenohypophysis were consistently reduced. Small doses of T4 (4 mug) induced significant TSH repletion in the pituitary within 2-6 h following injection. Accumulations of pituitary TSH to supranormal levels (15-fold increases) were achieved with 20 mug T4 at 6 and 24 h; higher doses (100-200 mug) inhibited the PTR at all time intervals tested (0.5-24 h). Administration of puromycin or actinomycin D did not influence the PTR. Protein content and labeled uridine uptake of the pituitary bore no apparent relationship to T4-induced TSH repletion in the gland. Blood clearance rate of exogenous rat TSH was measured prior to and during PTR. Plasma half-life was determined to be 13.6 and 19.9 min in euthyroid and chronically hypothyroid rats, respectively; it was not significantly altered from the latter during rebound (18.7 min). Calculations of theoretical TSH secretory rates prior to (50.5 +/- 4.4 mU/h) and after rebound with 20 mug T4 (25.4 +/- 4.2 mU/H) revealed that the reaccumulation of TSH in the pituitary induced with T4 cannot be attributed solely to inhibition of release, but may also involve enhancement of synthesis. It is concluded that T4 administration at high dose levels inhibits both synthesis and release of TSH from pituitary thyrotrophs, whereas low critical doses of T4 suppress release, but augment synthesis and/or facilitate conformational change in a pituitary precursor(s) molecule which renders it detectable by bioassay.
Assuntos
Bócio/metabolismo , Adeno-Hipófise/metabolismo , Hipófise/metabolismo , Glândula Tireoide/metabolismo , Tireotropina/metabolismo , Tiroxina/farmacologia , Animais , Dactinomicina/farmacologia , Feminino , Bócio/induzido quimicamente , Cinética , Taxa de Depuração Metabólica , Adeno-Hipófise/efeitos dos fármacos , Propiltiouracila , Puromicina/farmacologia , Ratos , Taxa Secretória , Tireotropina/sangue , Uridina/metabolismoRESUMO
Intravenous injection of the synthetic tripeptide (PyroGlu-His-Pro-NH2:TRH) effected the prompt release of TSH and prolactin (PRL) from the pituitary of the goitrous rat. Plasma TSH and PRL levels increased 2-3-fold within 1 min after the injection of 0.4 and 2 mug TRH. Intravenous injection of 20 mug of L-thyroxine (T4) induced repletion of TSH to supranormal levels in the adenohypophysis of goitrous rats without a significant change in PRL stores. The acute administration of TRH (2 and 50 mug) to rats after pituitary TSH rebound resulted in a simultaneous increase in circulating levels of the pituitary hormones; this was correlated with the prompt and vigorous extrusion of secretory granules from the pituitary cells. PRL content of the pituitary increased. A relationship was found in rats between the amount of TRH ingested in drinking water and plasma levels of PRL and TSH; hormonal stores in the adenohypophysis usually declined. Ingestion of large amounts of TRH (1,700 mug daily for 8 and 14 days) by the euthyroid rat resulted in a 2-3-fold elevation of the plasma TSH level. In PTU (propylthiouracil)-treated rats ingesting approximately the same amount of TRH, a plasma TSH increase failed to occur. The oral ingestion of TRH for 22-27 days by goitrous, TSH-rebounded rats resulted in a significant dimunution in the circulating levels of TSH and PRL, and in ultrastructural manifestations suggestive of impaired release by the adenohypophysis. It is concluded that the acute administration of TRH causes the rapid release of TSH and PRL from the pituitary of the chronically hypothyroid rat. The intensity of the response to TRH is enhanced after pituitary TSH rebound, and synthesis of PRL appears to be augmented. Chronic oral administration of TRH to the goitrous rat results in a diminished release of the pituitary hormones, despite ample stores in the gland.
Assuntos
Hipotireoidismo/tratamento farmacológico , Prolactina/metabolismo , Hormônio Liberador de Tireotropina/uso terapêutico , Tireotropina/metabolismo , Administração Oral , Animais , Feminino , Injeções Intravenosas , Adeno-Hipófise/análise , Ratos , Estimulação Química , Glândula Tireoide/ultraestrutura , Hormônio Liberador de Tireotropina/administração & dosagem , Hormônio Liberador de Tireotropina/farmacologia , Fatores de TempoRESUMO
Acute administration of PGE-1 in graded doses (0.25-1.0 mg) to pregnant rats in late gestation (21st day) induced significant release of TSH, prolactin and LH from the maternal, pituitary. Plasma levels of PRL and TSH were elevated (three- to fivefold) in cesarean sectioned mothers given 4-5 sc injections of PGE-1; higher doses also stimulated LH release. Comparable increases in blood TSH and PRL levels were also found in their newborn but no indication of enhanced hormone release from the fetal hypophysis was noted. Histologic alterations were observed in the ovary (luteal cell hypertrophy, cytopasmic clearing, nuclear condensation, sinusoidal distension) of mothers receiving PGE-1 treatment. Thyroidal radioiodine accumulation in mother, fetus and neonate were usually decreased whereas thyroid: blood radioactivity ratios were consistently incrased. It is concluded that PGE-1 treatment of th pregnant rat near term stimulates release of hormones from the pituitary in mothers (TSH, LH, PRL) and in their newborn (TSH, PRL). The results also strongly suggest that PGE-1 administration may induce morphologic and functional changes in target endocrines (ovary, thyroid) by direct and separate actions.