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1.
Oncogene ; 38(17): 3261-3273, 2019 04.
Article in English | MEDLINE | ID: mdl-30631150

ABSTRACT

Selective inhibition of BCL-2 is expected to enhance therapeutic vulnerability in luminal estrogen receptor-positive breast cancers. We show here that the BCL-2 dependency of luminal tumor cells is nevertheless mitigated by breast cancer-associated fibroblasts (bCAFs) in a manner that defines MCL-1 as another critical therapeutic target. bCAFs favor MCL-1 expression and apoptotic resistance in luminal cancer cells in a IL-6 dependent manner while their own, robust, survival also relies on MCL-1. Studies based on ex vivo cultures of human luminal breast cancer tissues further argue that the contribution of stroma-derived signals to MCL-1 expression shapes BCL-2 dependency. Thus, MCL-1 inhibitors are beneficial for targeted apoptosis of breast tumor ecosystems, even in a subtype where MCL-1 dependency is not intrinsically driven by oncogenic pathways.


Subject(s)
Breast Neoplasms/metabolism , Cancer-Associated Fibroblasts/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/deficiency , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Receptors, Estrogen/metabolism , Apoptosis/physiology , Breast Neoplasms/pathology , Cancer-Associated Fibroblasts/pathology , Cell Line, Tumor , Cell Survival/physiology , Female , Humans , Interleukin-6/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism
2.
Oncogenesis ; 6(10): e390, 2017 Oct 23.
Article in English | MEDLINE | ID: mdl-29058695

ABSTRACT

Breast cancer-associated fibroblasts (CAFs) have a crucial role in tumor initiation, metastasis and therapeutic resistance by secreting various growth factors, cytokines, protease and extracellular matrix components. Soluble factors secreted by CAFs are involved in many pathways including inflammation, metabolism, proliferation and epigenetic modulation, suggesting that CAF-dependent reprograming of cancer cells affects a large set of genes. This paracrine signaling has an important role in tumor progression, thus deciphering some of these processes could lead to relevant discoveries with subsequent clinical implications. Here, we investigated the mechanisms underlying the changes in gene expression patterns associated with the cross-talk between breast cancer cells and the stroma. From RNAseq data obtained from breast cancer cell lines grown in presence of CAF-secreted factors, we identified 372 upregulated genes, exhibiting an expression level positively correlated with the stromal content of breast cancer specimens. Furthermore, we observed that gene expression changes were not mediated through significant DNA methylation changes. Nevertheless, CAF-secreted factors but also stromal content of the tumors remarkably activated specific genes characterized by a DNA methylation pattern: hypermethylation at transcription start site and shore regions. Experimental approaches (inhibition of DNA methylation, knockdown of methyl-CpG-binding domain protein 2 and chromatin immunoprecipitation assays) indicated that this set of genes was epigenetically controlled. These data elucidate the importance of epigenetics marks in the cancer cell reprogramming induced by stromal cell and indicated that the interpreters of the DNA methylation signal have a major role in the response of the cancer cells to the microenvironment.

4.
Cell Death Dis ; 7: e2083, 2016 02 04.
Article in English | MEDLINE | ID: mdl-26844698

ABSTRACT

Proapoptotic molecules directly targeting the BCL-2 family network are promising anticancer therapeutics, but an understanding of the cellular stress signals that render them effective is still elusive. We show here that the tumor suppressor p53, at least in part by transcription independent mechanisms, contributes to cell death induction and full activation of BAX by BH3 mimetic inhibitors of BCL-xL. In addition to mildly facilitating the ability of compounds to derepress BAX from BCL-xL, p53 also provides a death signal downstream of anti-apoptotic proteins inhibition. This death signal cooperates with BH3-induced activation of BAX and it is independent from PUMA, as enhanced p53 can substitute for PUMA to promote BAX activation in response to BH3 mimetics. The acute sensitivity of mitochondrial priming to p53 revealed here is likely to be critical for the clinical use of BH3 mimetics.


Subject(s)
Apoptosis/drug effects , Biphenyl Compounds/pharmacology , Mitochondria/drug effects , Mitochondria/metabolism , Nitrophenols/pharmacology , Sulfonamides/pharmacology , Tumor Suppressor Protein p53/metabolism , bcl-X Protein/antagonists & inhibitors , Apoptosis/physiology , BH3 Interacting Domain Death Agonist Protein/metabolism , Biomimetic Materials/pharmacology , Cell Death/drug effects , Cell Death/physiology , Cell Line, Tumor , Cell Membrane Permeability/drug effects , HCT116 Cells , Humans , Piperazines/pharmacology , Tumor Suppressor Protein p53/genetics , bcl-2-Associated X Protein/metabolism
5.
Cell Death Dis ; 5: e1445, 2014 Oct 09.
Article in English | MEDLINE | ID: mdl-25299770

ABSTRACT

We sought to identify miRNAs that can efficiently induce apoptosis in ovarian cancer cells by overcoming BCL-X(L) and MCL1 anti-apoptotic activity, using combined computational and experimental approaches. We found that miR-491-5p efficiently induces apoptosis in IGROV1-R10 cells by directly inhibiting BCL-X(L) expression and by inducing BIM accumulation in its dephosphorylated form. This latter effect is due to direct targeting of epidermal growth factor receptor (EGFR) by miR-491-5p and consequent inhibition of downstream AKT and MAPK signalling pathways. Induction of apoptosis by miR-491-5p in this cell line is mimicked by a combination of EGFR inhibition together with a BH3-mimetic molecule. In contrast, SKOV3 cells treated with miR-491-5p maintain AKT and MAPK activity, do not induce BIM and do not undergo cell death despite BCL-XL and EGFR downregulation. In this cell line, sensitivity to miR-491-5p is restored by inhibition of both AKT and MAPK signalling pathways. Altogether, this work highlights the potential of miRNA functional studies to decipher cell signalling pathways or major regulatory hubs involved in cell survival to finally propose the rationale design of new strategies on the basis of pharmacological combinations.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis , Carcinoma/physiopathology , ErbB Receptors/genetics , ErbB Receptors/metabolism , Membrane Proteins/metabolism , MicroRNAs/metabolism , Ovarian Neoplasms/physiopathology , Proto-Oncogene Proteins/metabolism , bcl-X Protein/genetics , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11 , Carcinoma/genetics , Carcinoma/metabolism , Cell Line, Tumor , Down-Regulation , Female , Humans , Membrane Proteins/genetics , MicroRNAs/genetics , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Proto-Oncogene Proteins/genetics , bcl-X Protein/metabolism
6.
Cell Death Dis ; 5: e1291, 2014 Jun 12.
Article in English | MEDLINE | ID: mdl-24922075

ABSTRACT

Antimitotic agents such as microtubule inhibitors (paclitaxel) are widely used in cancer therapy while new agents blocking mitosis onset are currently in development. All these agents impose a prolonged mitotic arrest in cancer cells that relies on sustained activation of the spindle assembly checkpoint and may lead to subsequent cell death by incompletely understood molecular events. We have investigated the role played by anti-apoptotic Bcl-2 family members in the fate of mitotically arrested mammary tumor cells treated with paclitaxel, or depleted in Cdc20, the activator of the anaphase promoting complex. Under these conditions, a weak and delayed mitotic cell death occurs that is caspase- and Bax/Bak-independent. Moreover, BH3 profiling assays indicate that viable cells during mitotic arrest are primed to die by apoptosis and that Bcl-xL is required to maintain mitochondrial integrity. Consistently, Bcl-xL depletion, or treatment with its inhibitor ABT-737 (but not with the specific Bcl-2 inhibitor ABT-199), during mitotic arrest converts cell response to antimitotics to efficient caspase and Bax-dependent apoptosis. Apoptotic priming under conditions of mitotic arrest relies, at least in part, on the phosphorylation on serine 62 of Bcl-xL, which modulates its interaction with Bax and its sensitivity to ABT-737. The phospho-mimetic S62D-Bcl-xL mutant is indeed less efficient than the corresponding phospho-deficient S62A-Bcl-xL mutant in sequestrating Bax and in protecting cancer cells from mitotic cell death or yeast cells from Bax-induced growth inhibition. Our results provide a rationale for combining Bcl-xL targeting to antimitotic agents to improve clinical efficacy of antimitotic strategy in cancer therapy.


Subject(s)
Apoptosis , Breast Neoplasms/metabolism , Cell Cycle Checkpoints , bcl-X Protein/metabolism , Amino Acid Substitution , Biphenyl Compounds/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Humans , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/pathology , Mutation, Missense , Nitrophenols/pharmacology , Piperazines/pharmacology , Sulfonamides/pharmacology , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism , bcl-X Protein/genetics
7.
Cell Death Differ ; 20(5): 755-64, 2013 May.
Article in English | MEDLINE | ID: mdl-23429261

ABSTRACT

Although Bcl-2 family members control caspase activity by regulating mitochondrial permeability, caspases can, in turn, amplify the apoptotic process upstream of mitochondria by ill-characterized mechanisms. We herein show that treatment with a potent inhibitor of Bcl-2 and Bcl-xL, ABT-737, triggers caspase-dependent induction of the BH3-only protein, Mcl-1 inhibitor, Noxa. RNA interference experiments reveal that induction of Noxa, and subsequent cell death, rely not only on the transcription factor E2F-1 but also on its regulator pRb. In response to ABT-737, pRb is cleaved by caspases into a p68Rb form that still interacts with E2F-1. Moreover, pRb occupies the noxa promoter together with E2F-1, in a caspase-dependent manner upon ABT-737 treatment. Thus, caspases contribute to trigger the mitochondrial apoptotic pathway by coupling Bcl-2/Bcl-xL inhibition to that of Mcl-1, via the pRb/E2F-1-dependent induction of Noxa.


Subject(s)
Apoptosis/drug effects , Biphenyl Compounds/pharmacology , E2F1 Transcription Factor/metabolism , Nitrophenols/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Retinoblastoma Protein/metabolism , Sulfonamides/pharmacology , Apoptosis Regulatory Proteins/genetics , Breast Neoplasms , Caspases/metabolism , Cell Line, Tumor , DNA-Binding Proteins/genetics , E2F1 Transcription Factor/genetics , Female , Humans , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins , Myeloid Cell Leukemia Sequence 1 Protein , Nuclear Proteins/genetics , Piperazines/pharmacology , Promoter Regions, Genetic , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , RNA Interference , RNA, Small Interfering , Retinoblastoma Protein/genetics , Transcription, Genetic , Tumor Protein p73 , Tumor Suppressor Proteins/genetics , bcl-2-Associated X Protein/antagonists & inhibitors , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism , bcl-X Protein/antagonists & inhibitors , bcl-X Protein/genetics , bcl-X Protein/metabolism
8.
Mol Cell Biol ; 31(4): 832-44, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21173168

ABSTRACT

Bcl-2 homologues (such as Bcl-x(L)) promote survival in part through sequestration of "activator" BH3-only proteins (such as Puma), preventing them from directly activating Bax. It is thus assumed that inhibition of interactions between activators and Bcl-x(L) is a prerequisite for small molecules to antagonize Bcl-x(L) and induce cell death. The biological properties, described here of a terphenyl-based alpha-helical peptidomimetic inhibitor of Bcl-x(L) attest that displacement of Bax from Bcl-x(L) is also critical. Terphenyl 14 triggers Bax-dependent but Puma-independent cell death, disrupting Bax/Bcl-x(L) interactions without affecting Puma/Bcl-x(L) interactions. In cell-free assays, binding of inactive Bax to Bcl-x(L), followed by its displacement from Bcl-x(L) by terphenyl 14, produces mitochondrially permeabilizing Bax molecules. Moreover, the peptidomimetic kills yeast cells that express Bax and Bcl-x(L), and it uses Bax-binding Bcl-x(L) to induce mammalian cell death. Likewise, ectopic expression of Bax in yeast and mammalian cells enhances sensitivity to another Bcl-x(L) inhibitor, ABT-737, when Bcl-x(L) is present. Thus, the interaction of Bcl-x(L) with Bax paradoxically primes Bax at the same time it keeps Bax activity in check, and displacement of Bax from Bcl-x(L) triggers an apoptotic signal by itself. This mechanism might contribute to the clinical efficiency of Bcl-x(L) inhibitors.


Subject(s)
bcl-2-Associated X Protein/metabolism , bcl-X Protein/metabolism , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Binding Sites , Biphenyl Compounds/pharmacology , Cell Death/drug effects , Cell Death/genetics , Cell Death/physiology , Cell Line, Tumor , Cell-Free System , Cells, Cultured , Gene Knockdown Techniques , HeLa Cells , Humans , In Vitro Techniques , Mice , Mice, Knockout , Mitochondria/metabolism , Models, Biological , Molecular Mimicry , Molecular Sequence Data , Mutation , Nitrophenols/pharmacology , Piperazines/pharmacology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA, Small Interfering/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sulfonamides/pharmacology , Terphenyl Compounds/pharmacology , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Two-Hybrid System Techniques , bcl-2-Associated X Protein/deficiency , bcl-2-Associated X Protein/genetics , bcl-X Protein/deficiency , bcl-X Protein/genetics
9.
J Control Release ; 151(1): 74-82, 2011 Apr 10.
Article in English | MEDLINE | ID: mdl-21138749

ABSTRACT

Taking advantage from the development of SV30, a new analogue of the pro-apoptotic molecule HA14-1, the aim of this study was to functionally evaluate SV30 and to develop safe nanocarriers for its administration. By using an inversion phase process, 57nm organic solvent-free lipid nanocapsules loaded with SV30 (SV30-LNCs) were formulated. Biological performance of SV30 and SV30-LNCs were evaluated on F98 cells that express Bax and Bcl-2, through survival assays, HPLC, flow cytometry, confocal microscopy and spectral imaging. We observed that SV30 alone or in combination with paclitaxel, etoposide or beam radiation could trigger cell death in a similar fashion to HA14-1. Although partially blocked by Z-VAD-fmk, this effect was coincident to caspase-3 activation. Hence, we established that SV30-LNCs improved SV30 biological activity together with a potentiation of the mitochondrial membrane potential decrease. Interestingly, flow cytometry and confocal analysis indicated that SV30 itself conferred to LNCs improved mitochondrial targeting skills that may present a great interest toward the development of mitochondria targeted nanomedicines.


Subject(s)
Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Benzopyrans/chemistry , Glioma/drug therapy , Lipids/chemistry , Mitochondria/metabolism , Nanocapsules/chemistry , Nitriles/chemistry , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Caspase 3/metabolism , Cell Line, Tumor , Mitochondria/drug effects , Rats
10.
Cancer Gene Ther ; 16(6): 498-507, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19165235

ABSTRACT

Cetuximab (Erbitux) is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody whose activity is related to the inhibition of EGFR downstream signaling pathways. P53 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) have been reported to control the functionality of PI3K/AKT signaling. In this study we evaluated whether reintroducing P53 using non-viral gene transfer enhances PTEN-mediated inhibition of PI3K/AKT signaling by cetuximab in PC3 prostate adenocarcinoma cell line bearing p53 and pten mutations. Signaling phosphoproteins expression was analyzed using Bio-Plex phosphoprotein array and western blot. Apoptosis induction was evaluated from BAX expression, caspase-3 activation and DNA fragmentation analyses. The results presented show that p53 and pten gene transfer additionally mediated cell growth inhibition and apoptosis induction by restoral of signaling functionality, which enabled the control of PI3K/AKT and MAPKinase signaling pathways by cetuximab in PC3 cells. These results highlight the interest of the analysis of signaling phosphoproteins expression as molecular predictive markers for response to cetuximab and show that p53 and pten mutations could be key determinants of cell response to cetuximab through the functional impact of these mutations on cell signaling.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , ErbB Receptors/antagonists & inhibitors , PTEN Phosphohydrolase/genetics , Signal Transduction , Tumor Suppressor Protein p53/genetics , Antibodies, Monoclonal, Humanized , Apoptosis , Cell Line, Tumor , Cetuximab , ErbB Receptors/genetics , ErbB Receptors/metabolism , Humans , Immunohistochemistry , Male , PTEN Phosphohydrolase/metabolism , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Transfection , Tumor Suppressor Protein p53/metabolism
11.
Br J Cancer ; 97(7): 883-7, 2007 Oct 08.
Article in English | MEDLINE | ID: mdl-17876327

ABSTRACT

The aim of this study was to evaluate with a long follow-up the efficacy of concomitant chemoradiotherapy in non-metastatic inflammatory breast cancer (IBC) and to evaluate the breast conservation rate. Between 1990 and 2000, 66 non-metastatic patients with IBC were treated with chemotherapy and concomitant irradiation. The induction chemotherapy consisted of epirubicine, cyclophosphamide and vindesine, in association with split-course bi-fractionated irradiation to a total dose of 65 Gy with concomitant cisplatin and 5-fluorouracil. Maintenance chemotherapy consisted of high-dose methotrexate and six cycles of epirubicine, cyclophosphamide and fluorouracil. Hormonal treatment was given if indicated. Mastectomy was not systemic. Among 65 evaluable patients, 57 (87.6%) achieved a complete clinical response and had a breast conservation. Only six loco regional relapses were noted in six patients with a delay of 20 months and with concomitant metastatic dissemination in four cases. Median disease-free survival (DFS) was 28 months. Median overall survival (OS) was 63 months and median follow-up was 55.5 months. Induction chemotherapy and concomitant irradiation is feasible in patients with IBC, permitting a breast conservation with a high rate of local control with an OS comparable to that of the best recent series.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/radiotherapy , Adult , Aged , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/drug therapy , Carcinoma, Ductal, Breast/pathology , Carcinoma, Ductal, Breast/radiotherapy , Carcinoma, Lobular/drug therapy , Carcinoma, Lobular/pathology , Carcinoma, Lobular/radiotherapy , Cisplatin/administration & dosage , Combined Modality Therapy , Cyclophosphamide/administration & dosage , Disease-Free Survival , Dose Fractionation, Radiation , Epirubicin/administration & dosage , Female , Fluorouracil/administration & dosage , Follow-Up Studies , Humans , Menopause , Middle Aged , Remission Induction , Survival Rate , Time Factors , Treatment Outcome
12.
Oncogene ; 26(46): 6653-64, 2007 Oct 11.
Article in English | MEDLINE | ID: mdl-17471233

ABSTRACT

Oncostatin M (OSM), a cytokine of the interleukin-6 family, induces growth arrest and differentiation of osteoblastic cells into glial-like/osteocytic cells. Here, we asked whether OSM regulates apoptosis of normal or transformed (osteosarcoma) osteoblasts. We show that OSM sensitizes cells to apoptosis induced by various death inducers such as staurosporine, ultraviolet or tumor necrosis factor-alpha. Apoptosis is mediated by the mitochondrial pathway, with release of cytochrome c from the mitochondria to the cytosol and activation of caspases-9 and -3. DNA micro-arrays revealed that OSM modulates the expression of Bax, Bad, Bnip3, Bcl-2 and Mcl-1. Pharmacological inhibitors, dominant-negative signal transducer and activator of transcriptions (STATs), stable RNA interference and knockout cells indicated that the transcription factors p53 and STAT5, which are activated by OSM, are implicated in the sensitization to apoptosis, being responsible for Bax induction and Bcl-2 reduction, respectively. These results indicate that, in addition to growth arrest and induced differentiation, OSM also sensitizes normal and transformed osteoblasts to apoptosis by a mechanism implicating (i) activation and nuclear translocation of STAT5 and p53 and (ii) an increased Bax/Bcl-2 ratio. Therefore, association of OSM with kinase inhibitors such as Sts represents new therapeutic opportunities for wild-type p53 osteosarcoma.


Subject(s)
Apoptosis , Oncostatin M/pharmacology , Apoptosis/drug effects , Carbazoles/pharmacology , Caspases/metabolism , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Indole Alkaloids/pharmacology , Osteosarcoma , Proto-Oncogene Proteins c-bcl-2/metabolism , STAT5 Transcription Factor , Tumor Suppressor Protein p53 , bcl-2-Associated X Protein/metabolism
14.
Cell Death Differ ; 14(4): 785-94, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17096026

ABSTRACT

The association of Bax with mitochondria is an essential step in the implementation of apoptosis. By using a bacterial two-hybrid assay and crosslinking strategies, we have identified TOM22, a component of the translocase of the outer mitochondrial membrane (TOM), as a mitochondrial receptor of Bax. Peptide mapping showed that the interaction of Bax with TOM22 involved the first alpha helix of Bax and possibly two central alpha helices, which are homologous to the pore forming domains of some toxins. Antibodies directed against TOM22 or an antisense knockdown of the expression of TOM22 specifically inhibited the association of Bax with mitochondria and prevented Bax-dependent apoptosis. In yeast, a haploid strain for TOM22 exhibited a decreased expression of TOM22 and mitochondrial association of ectopically expressed human Bax. Our data provide a new perspective on the mechanism of association of Bax with mitochondria as it involves a classical import pathway.


Subject(s)
Membrane Transport Proteins/metabolism , Mitochondria, Liver/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Voltage-Dependent Anion Channels/metabolism , bcl-2-Associated X Protein/metabolism , Animals , Apoptosis/physiology , Gene Silencing , Glioblastoma , Glioma , Humans , Microscopy, Confocal , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/metabolism , Protein Binding/physiology , Protein Transport/physiology , Rats , Receptors, Cell Surface , Receptors, Cytoplasmic and Nuclear/metabolism , Tumor Cells, Cultured , bcl-2-Associated X Protein/deficiency
15.
Oncology ; 67(3-4): 257-61, 2004.
Article in English | MEDLINE | ID: mdl-15557787

ABSTRACT

OBJECTIVE: A phase I/II study was carried out to determine the recommended dose (RD) and to assess the efficacy and safety of a weekly docetaxel-vinorelbine combination in advanced breast cancer (ABC) patients. METHODS: Twenty-four female patients with histologically proven ABC received intravenous vinorelbine (20 min) followed by intravenous docetaxel (1 h) on days 1, 8 and 15 of a 4-week cycle. Starting doses were 20 mg/m2 docetaxel and 15 mg/m2 vinorelbine. RESULTS: Patients had a median age of 62 years (range 38-74 years), and 92% had performance status 0-1. The most common sites of metastases were the lungs (32%), liver (29%) and bone (14%). Seventy-one percent of patients had received prior chemotherapy. The RDs of docetaxel and vinorelbine were 20 and 15 mg/m2, respectively. Dose-limiting toxicities were neutropenia-induced dose delay and febrile neutropenia. The response rate at the RD was 43%. All responses were seen in non-pretreated patients. Grade 3-4 neutropenia occurred in 80% of patients, 3 of whom experienced febrile neutropenia and died as a possible consequence of neutropenia. CONCLUSION: This docetaxel-vinorelbine combination as first-line therapy yields a response rate similar to that of single-agent docetaxel as second-line therapy. However, given the high rate of myelotoxicity, higher doses are not feasible.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Vinblastine/analogs & derivatives , Adult , Aged , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Docetaxel , Drug Administration Schedule , Female , Humans , Middle Aged , Taxoids/administration & dosage , Treatment Outcome , Vinblastine/administration & dosage , Vinorelbine
16.
Ann Oncol ; 15(7): 1018-24, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15205194

ABSTRACT

PURPOSE: To determine maximum tolerated dose, safety and efficacy of weekly 24 h infusional 5-fluorouracil (5-FU) combined alternately with oxaliplatin and irinotecan. PATIENTS AND METHODS: Advanced colorectal carcinoma patients in first- or second-line chemotherapy received increasing doses of 5-FU (weekly 24 h continuous intravenous infusion without leucovorin) on days 1, 8, 15 and 22, irinotecan days 1 and 15; and oxaliplatin days 8 and 22, every 35 days. RESULTS: Thirty-four patients received 175 cycles. The median age was 64 years (range 47-78). Eighteen per cent of patients had the primary tumor in the rectum, with a median of one disease site (range one to three), and liver involvement in 88% and lung in 38%. Six (18%) patients had chemotherapy for prior advanced disease. The most frequent grade 3-4 toxicity was neutropenia (41% of patients), but the regimen was well tolerated clinically, with febrile neutropenia in two patients and grade 4 neutropenia lasting >7 days in one; grade 3-4 diarrhea, nausea and vomiting in 6% of patients; grade 3-4 peripheral neuropathy in 9% of patients. Seventeen patients had a partial response (50%; 95% confidence interval 33%-67%), 13 had stable disease and one had progressive disease. Five patients underwent metastatic surgical resection after tumor shrinkage. Median response duration was 14 months (range 4.7-29.2+) and median time to progression was 11.3 months (range 1.1+-30.7+). CONCLUSIONS: This combination three-drug regimen is feasible and well tolerated without toxicity overlap. Preliminary antitumor activity compares well with standard double combinations, with an unusually long median time to progression. The recommended dose is 5-FU 3000 mg/m(2), weekly for 4 weeks, irinotecan 100 mg/m(2) days 1 and 15, oxaliplatin 80 mg/m(2) days 8 and 22. Further assessment of antitumor activity and safety is warranted.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Camptothecin/analogs & derivatives , Colorectal Neoplasms/drug therapy , Aged , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Camptothecin/administration & dosage , Camptothecin/adverse effects , Camptothecin/therapeutic use , Diarrhea/chemically induced , Dose-Response Relationship, Drug , Fluorouracil/administration & dosage , Fluorouracil/adverse effects , Fluorouracil/therapeutic use , Humans , Infusion Pumps , Irinotecan , Middle Aged , Nausea/chemically induced , Neutropenia/chemically induced , Organoplatinum Compounds/administration & dosage , Organoplatinum Compounds/adverse effects , Organoplatinum Compounds/therapeutic use , Oxaliplatin , Time Factors , Treatment Outcome
18.
Nat Cell Biol ; 2(3): 156-62, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10707086

ABSTRACT

Release of cytochrome c from mitochondria triggers activation of caspase proteases and death of a cell by apoptosis. However, the mechanism and kinetics of cytochrome c release remain unknown. Here we study this event by using green fluorescent protein (GFP)-tagged cytochrome c, and find that the release of cytochrome-c-GFP always precedes exposure of phosphatidylserine and the loss of plasma-membrane integrity - characteristics of apoptotic cells. Once initiated, the release of cytochrome- c-GFP continues until all of the protein is released from all mitochondria in individual cells, within about 5 minutes, regardless of the type or strength of stimulus or the time elapsed since the stimulus was applied. Temperatures ranging from 24 degrees C to 37 degrees C do not change the duration of release, and nor does the addition of caspase inhibitors. Further, we find that the electron-transport chain can maintain the mitochondrial transmembrane potential even after cytochrome c has been released.


Subject(s)
Apoptosis , Cytochrome c Group/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Caspase Inhibitors , Caspases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Cytochrome c Group/genetics , Digitonin/pharmacology , Electron Transport/drug effects , Electron Transport/radiation effects , Enzyme Activation/drug effects , Enzyme Activation/radiation effects , Enzyme Inhibitors/pharmacology , Green Fluorescent Proteins , HeLa Cells , Humans , Image Processing, Computer-Assisted , Intracellular Membranes/drug effects , Intracellular Membranes/radiation effects , Luminescent Proteins/genetics , Membrane Potentials/drug effects , Mitochondria/metabolism , Oligomycins/pharmacology , Phosphatidylserines/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sodium Azide/pharmacology , Temperature , Ultraviolet Rays
19.
Biochem Biophys Res Commun ; 260(3): 582-91, 1999 Jul 14.
Article in English | MEDLINE | ID: mdl-10403809

ABSTRACT

It has been suggested that BCL-2 family members associate with certain organelles through their hydrophobic C-terminus which in the case of bcl-2, appears to play a key role in the regulation of apoptosis. We have investigated the association of bax with microsomal, nuclear and mitochondrial membranes using a cell-free system and found, contrary to bcl-2, that bax binds poorly to these organelles. Deletion of the C-terminal of bax (baxDeltaC) or exchanging the C-terminal ends of bax and bcl-XL suggests that the bax C-terminus is not an addressing/anchoring signal. In agreement with this observation, HL-60 cells transfected with either bax or baxDeltaC show no difference in sensitivity to an apoptotic signal. In the cell-free system, at low pH, bax becomes associated with mitochondria after a change of conformation, a result consistant with its structural homology with certain bacterial toxins. In HL-60 cells, as observed in the cell-free system, bax acquired a protease resistant conformation upon its translocation from the cytosol to the mitochondria after the induction of apoptosis.


Subject(s)
Cell Membrane/metabolism , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Amino Acid Sequence , Animals , Apoptosis/drug effects , Cell Membrane/drug effects , Cell Nucleus/metabolism , Cytosol/drug effects , Cytosol/metabolism , Dogs , Doxorubicin/pharmacology , HL-60 Cells , Humans , Hydrogen-Ion Concentration , Liver/cytology , Microsomes/metabolism , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Molecular Sequence Data , Pancreas/cytology , Protein Binding/drug effects , Protein Conformation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Deletion , bcl-2-Associated X Protein
20.
Genes Dev ; 13(11): 1367-81, 1999 Jun 01.
Article in English | MEDLINE | ID: mdl-10364155

ABSTRACT

Expression of c-Myc sensitizes cells to a wide range of pro-apoptotic stimuli. We here show that this pro-apoptotic effect is mediated through release of mitochondrial holocytochrome c into the cytosol. First, activation of c-Myc triggers release of cytochrome c from mitochondria. This release is caspase-independent and blocked by the survival factor IGF-1. Second, c-Myc-induced apoptosis is blocked by microinjection of anticytochrome c antibody. In addition, we show that microinjection of holocytochrome c mimics the effect of c-Myc activation, sensitizing cells to DNA damage and to the CD95 pathway. Both p53 and CD95/Fas signaling have been implicated in c-Myc-induced apoptosis but neither was required for c-Myc-induced cytochrome c release. Nonetheless, inhibition of CD95 signaling in fibroblasts did prevent c-Myc-induced apoptosis, apparently by obstructing the ability of cytosolic cytochrome c to activate caspases. We conclude that c-Myc promotes apoptosis by causing the release of cytochrome c, but the ability of cytochrome c to activate apoptosis is critically dependent upon other signals.


Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis , Cytochrome c Group/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Signal Transduction , Animals , Antibodies, Monoclonal/metabolism , Carrier Proteins/metabolism , Caspases/metabolism , Cell Line , Cytosol/metabolism , Enzyme Activation , Fas-Associated Death Domain Protein , Microinjections , Mitochondria/metabolism , Rats , Tumor Suppressor Protein p53/metabolism , X-Rays , fas Receptor/metabolism
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