ABSTRACT
PURPOSE: Intravenously (i.v.) administered nanomedicines have the potential for tumour targeting due to the enhanced permeability and retention (EPR) effect, but in vivo tumour models are rarely calibrated with respect to functional vascular permeability and/or mechanisms controlling intratumoural drug release. Here the effect of tumour type and tumour size on EPR-mediated tumour localisation and cathepsin B-mediated drug release was studied. METHODS: Evans Blue (10 mg/kg) and an N-(2-hydroxypropyl) methacrylamide (HPMA) copolymerdoxorubicin (Dox) conjugate (FCE28068) (5 mg/kg Dox-equiv) were used as probes and tumour levels (and Dox release) measured at 1 h after i.v. administration in a panel of murine and human xenograft tumours. RESULTS: Evans Blue and FCE28068 displayed similar tumour levels in the range of 218 % dose/g at 1 h for B16F10 and L1210. Approximately half of the tumour models evaluated exhibited tumour size-dependent accumulation of FCE28068; smaller tumours had the highest accumulation. Administration of free Dox (5 mg/kg) produced tumour levels of \2.5 % dose/g independent of tumour size. Whereas the degree of EPR-mediated targeting showed *12-fold difference across the tumour models evaluated, Dox release from FCE28068 at 1 h displayed *200-fold variation. CONCLUSIONS: Marked heterogeneity was seen in terms of EPR effect and Dox release rate, underlining the need to carefully calibrate tumour models used to benchmark nanomedicines against known relevant standard agents and for optimal development of strategies for late pre-clinical and clinical development.
Subject(s)
Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Cathepsin B/pharmacology , Disease Models, Animal , Nanotechnology , Acrylamides/administration & dosage , Acrylamides/pharmacokinetics , Animals , Antibiotics, Antineoplastic/pharmacokinetics , Antibiotics, Antineoplastic/pharmacology , Antibiotics, Antineoplastic/therapeutic use , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Coloring Agents , Delayed-Action Preparations , Doxorubicin/administration & dosage , Doxorubicin/pharmacokinetics , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Evans Blue , Humans , Leukemia L1210/drug therapy , Mice , Permeability , Polyglutamic Acid , Polymers , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Lymphomas frequently retain wild-type (wt) p53 function but overexpress HDM2, thereby compromising p53 activity. Therefore, lymphoma is a suitable model for studying the therapeutic value of disrupting the HDM2-p53 interaction by small-molecule inhibitors (SMIs). HDM2 have been developed and are under various stages of preclinical and clinical investigation. Previously, we examined the anti-lymphoma activity of MI-319, the laboratory grade of a new class of HDM2 SMI, the spiro-oxindole, in follicular lymphoma. Since then, MI-219, the clinical grade has become readily available. This study further examines the preclinical effects and mechanisms of MI-219 in a panel of human lymphoma cell lines as well as a cohort of patient-derived B-lymphocytes for its potential clinical use. RESULTS: Preclinical assessment of MI-219 was evaluated by means of an in vitro and ex vivo approach and compared to Nutlin-3, the gold standard. Characterization of p53 activity and stability were assessed by quantitative PCR, Western blot, and immunoprecipitation. Biological outcome was measured using Trypan blue exclusion assay, Annexin V/PI, PARP and caspase-3 cleavage. Surprisingly, the overall biological effects of Nutlin-3 were more delayed (48 h) while MI-219 triggered an earlier response (12-24 h), predominantly in the form of apoptotic cell death. Using a cell free autoubiquitination assay, neither agent interfered with HDM2 E3 ligase function. MI-219 was more effective in upregulating wt-p53 stabilization compared to Nutlin-3. MI-219, but not Nutlin-3, enhanced the autoubiquitination and degradation of HDM2. CONCLUSIONS: Our data reveals unexpected differences between MI-219 and the well-studied Nutlin-3 in lymphoma cell lines and patient samples. We suggest a novel mechanism for MI-219 that alters the functional activity of HDM2 through enhanced autoubiquitination and degradation. Additionally, this mechanism appears to correspond to biological outcome. Our results provide evidence that different classes of HDM2 SMIs elicit molecular events that extend beyond HDM2-p53 dissociation which may be of biological and potentially therapeutic importance.
Subject(s)
Imidazoles/pharmacology , Indoles/pharmacology , Lymphoma, B-Cell/drug therapy , Lymphoma, B-Cell/metabolism , Piperazines/pharmacology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Spiro Compounds/pharmacology , Tumor Suppressor Protein p53/metabolism , Aged , Aged, 80 and over , Apoptosis/drug effects , Cell Line, Tumor , Female , Humans , Lymphoma, B-Cell/pathology , Male , Middle Aged , Proto-Oncogene Proteins c-mdm2/metabolism , Ubiquitination/drug effectsABSTRACT
BACKGROUND: Pemetrexed has emerged as one of the most active agents for the treatment of patients with advanced non-small cell lung cancer (NSCLC). We conducted a phase II study to assess the efficacy and feasibility of integrating pemetrexed in a concurrent therapy plan for patients with stage III NSCLC. METHODS: Patients with stage III NSCLC with performance status 0 to 1, adequate organ function including pulmonary function, and V20 less than 40% were eligible. Patients were treated with cisplatin 75 mg/m² (first five patients 60 mg/m²) and pemetrexed 500 mg/m² every 21 days for three cycles with chest radiotherapy to 66 Gy. Patients then received three cycles of docetaxel 75 mg/m² every 21 days. Tumors were analyzed for Excision Repair Cross Complementation Group 1 and thymidylate synthase. RESULTS: Patient characteristics (N = 28) were median age, 60; males, 68%; stage IIIB, 64%; and squamous cell, 43%. Twenty-four patients (86%) completed all three cycles of cisplatin/pemetrexed. Of the 24 patients eligible for docetaxel, 21 (87%) received it. Grade 3/4 toxicities were neutropenia (39%), febrile neutropenia (14%), esophagitis (14%), and pneumonitis (4%). Median survival was 34 months, and 1-year survival was 66%. Survival was not significantly different in squamous and other histology patients. Tumor analysis in 16 patients showed that moderate/strong expression of thymidylate synthase was significantly associated with progression-free survival and overall survival. CONCLUSION: Integrating pemetrexed in a concurrent therapy regimen for patients with stage III NSCLC is feasible and was associated with a median survival of 34 months.
Subject(s)
Adenocarcinoma/therapy , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Large Cell/therapy , Carcinoma, Non-Small-Cell Lung/therapy , Carcinoma, Squamous Cell/therapy , Lung Neoplasms/therapy , Adenocarcinoma/drug therapy , Adenocarcinoma/radiotherapy , Adult , Aged , Aged, 80 and over , Carcinoma, Large Cell/drug therapy , Carcinoma, Large Cell/radiotherapy , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/radiotherapy , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/radiotherapy , Cisplatin/administration & dosage , Combined Modality Therapy , DNA-Binding Proteins/metabolism , Docetaxel , Endonucleases/metabolism , Feasibility Studies , Female , Follow-Up Studies , Glutamates/administration & dosage , Guanine/administration & dosage , Guanine/analogs & derivatives , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/radiotherapy , Male , Middle Aged , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/radiotherapy , Neoplasm Recurrence, Local/therapy , Neoplasm Staging , Pemetrexed , Survival Rate , Taxoids/administration & dosage , Thymidylate Synthase/metabolism , Treatment OutcomeABSTRACT
The BCA2 protein contains a RING H2 finger and a Zn finger near the N-terminus and has E3 ligase activity. RING finger proteins play critical roles in mediating the transfer of ubiquitin and ubiquitin like modifiers to heterologous substrates as well as to the RING finger proteins themselves. Protein modification by ubiquitin and small ubiquitin-related modifier (SUMO) plays a pivotal role in protein homeostasis and is critical to regulating basic cellular processes such as proliferation, differentiation, apoptosis, intracellular signaling, and gene-transcriptional regulation. The addition of ubiquitin or SUMO can modulate the ability of proteins to interact with their partners, alter their patterns of sub-cellular localization and control their stability. It is clear that SUMO influences many different biological processes however recent data suggest that it is specifically important in the regulation of transcription. BCA2 is an E3 ligase that interacts with the SUMO conjugating enzyme Ubc9. It could therefore function as an E3 in the sumoylation of various transcription factors. We have found that the BCA2 is co-expressed with the estrogen receptor in 74% of ER-positive invasive ductal carcinomas from a 635 member breast cancer cohort (p = 0.004). At the cellular level, BCA2 co-localizes with ER and it appears that at the transcriptional level BCA2 mRNA expression is regulated by estrogen. Bioinformatic analysis of the BCA2 promoter region revealed ER and PR binding sites as well as that of other more general transcription factors. The data presented here provides an overview of the potential involvement of the BCA2 in hormone responsive breast cancer and opens up avenues that should be exploited to better understand the regulation of ER expression, growth of breast cancer cells, and the importance of BCA2.
ABSTRACT
Aminoflavone (AF), the active component of a novel anticancer agent (AFP464) in phase I clinical trials, is a ligand of the aryl hydrocarbon receptor (AhR). AhR dimerizes with HIF-1beta/AhR, which is shared with HIF-1alpha, a transcription factor critical for the response of cells to oxygen deprivation. To address whether pharmacologic activation of the AhR pathway might be a potential mechanism for inhibition of HIF-1, we tested the effects of AF on HIF-1 expression. AF inhibited HIF-1alpha transcriptional activity and protein accumulation in MCF-7 cells. However, inhibition of HIF-1alpha by AF was independent from a functional AhR pathway. Indeed, AF inhibited HIF-1alpha expression in Ah(R100) cells, in which the AhR pathway is functionally impaired, yet did not induce cytotoxicity, providing evidence that these effects are mediated by distinct signaling pathways. Moreover, AF was inactive in MDA-MB-231 cells, yet inhibited HIF-1alpha in MDA-MB-231 cells transfected with the SULT1A1 gene. AF inhibited HIF-1alpha mRNA expression by approximately 50%. Notably, actinomycin-D completely abrogated the ability of AF to downregulate HIF-1alpha mRNA, indicating that active transcription was required for the inhibition of HIF-1alpha expression. Finally, AF inhibited HIF-1alpha protein accumulation and the expression of HIF-1 target genes in MCF-7 xenografts. These results show that AF inhibits HIF-1alpha in an AhR-independent fashion, and they unveil additional activities of AF that may be relevant for its further clinical development.
Subject(s)
Breast Neoplasms/drug therapy , Flavonoids/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Receptors, Aryl Hydrocarbon/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/antagonists & inhibitors , Basic Helix-Loop-Helix Transcription Factors/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , DNA Damage , Female , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Ligands , Mice , Mice, Nude , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Transcription, Genetic/drug effects , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Heat shock protein (Hsp) 90 inhibition affects the Raf kinase signaling pathway and could enhance antitumor effects of sorafenib, a Raf kinase inhibitor. The combination of sorafenib and tanespimycin [17-allyl-amino-geldanamycin (17-AAG); NSC 330507/KOS-953] was evaluated in a phase I trial with the primary objective of defining a phase II dose. PATIENTS AND METHODS: The dose cohorts consisted of fixed continuous oral dosing of 400 mg sorafenib twice daily, starting at 14 days before tanespimycin, which was administered intravenously at escalating doses (starting at 300 mg/m,(2) with 50 mg/m(2) increments), on days 1, 8, and 15 in a 28-day cycle. Toxicity was assessed weekly, and response was evaluated every two cycles. RESULTS: Twenty-seven toxicity-evaluable patients were enrolled and treated at four dose levels. Predominant primary malignancies were renal cancer (12), melanoma (6), and colorectal cancer (4). Dose-limiting toxicities of grade 4 transaminitis and grade 3 hand-foot syndrome in one patient each were observed at 450 mg/m(2) of tanespimycin. One hundred fourteen cycles were administered with a median of four cycles (range 1-17 cycles). Plasma concentrations of sorafenib and metabolites reached steady state after 7 days. Tanespimycin did not alter sorafenib concentrations. Pharmacodynamics showed a decrease in Hsp90 levels and induction of Hsp70. Clinical efficacy was observed in 9 of 12 renal cancer patients and 4 of 6 melanoma patients CONCLUSIONS: Recommended phase II doses of this combination are 400 mg sorafenib twice daily and 400 mg/m(2) tanespimycin on days 1, 8, and 15, every 28 days. Clinical and pharmacodynamic activity was observed in kidney cancer and melanoma.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Benzenesulfonates , Benzoquinones , Colorectal Neoplasms/drug therapy , Kidney Neoplasms/drug therapy , Lactams, Macrocyclic , Melanoma/drug therapy , Pyridines , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Antineoplastic Combined Chemotherapy Protocols/pharmacokinetics , Benzenesulfonates/administration & dosage , Benzenesulfonates/adverse effects , Benzenesulfonates/pharmacokinetics , Benzoquinones/administration & dosage , Benzoquinones/adverse effects , Benzoquinones/pharmacokinetics , Clinical Trials, Phase II as Topic , Cohort Studies , Drug Administration Schedule , Drug-Related Side Effects and Adverse Reactions , Female , Follow-Up Studies , Humans , Lactams, Macrocyclic/administration & dosage , Lactams, Macrocyclic/adverse effects , Lactams, Macrocyclic/pharmacokinetics , Male , Middle Aged , Niacinamide/analogs & derivatives , Phenylurea Compounds , Pyridines/administration & dosage , Pyridines/adverse effects , Pyridines/pharmacokinetics , Sorafenib , Survival AnalysisABSTRACT
Human E1 is a key player in protein ubiquitination, however the E1 structure is not available. In this paper, we describe the derivation of a human E1 structure using molecular modelling based on the crystal structure of S. cerevisiae E1 and M. Musculus E1. Key interactions between our E1 model and ubiquitin are also discussed.
ABSTRACT
Poly(ADP-ribose) polymerase inhibitors are an exciting new class of antineoplastic agents that impair the ability of cells to recover from DNA damage. They are most effective in the setting of inherent DNA repair defects, such as in cancers resulting from BRCA gene mutations, or in the setting of DNA-damaging chemotherapeutic agents. This article reviews the background and development of these agents in the laboratory, as well as the rationale for the biologic correlative studies used in clinical trials. The most recent data from the clinical trials of olaparib (AZD2281, KU-0059436), BSI-201, AG014699, ABT-888, and INO-1001 and descriptions of ongoing studies are also presented.
Subject(s)
Antineoplastic Agents/therapeutic use , Neoplasms/drug therapy , Poly(ADP-ribose) Polymerase Inhibitors , Animals , Benzimidazoles/therapeutic use , Clinical Trials as Topic , Humans , Indoles/therapeutic use , Phthalazines/therapeutic use , Piperazines/therapeutic useABSTRACT
The zinc-ejecting aldehyde dehydrogenase (ALDH) inhibitory drug disulfiram (DSF) was found to be a breast cancer-associated protein 2 (BCA2) inhibitor with potent antitumor activity. We herein describe our work in the synthesis and evaluation of new series of zinc-affinic molecules to explore the structural requirements for selective BCA2-inhibitory antitumor activity. An N(C=S)S-S motif was found to be required, based on selective activity in BCA2-expressing breast cancer cell lines and against recombinant BCA2 protein. Notably, the DSF analogs (3a and 3c) and dithio(peroxo)thioate compounds (5d and 5f) were found to have potent activity (submicromolar IC(50)) in BCA2 positive MCF-7 and T47D cells but were inactive (IC(50) > 10 microM) in BCA2 negative MDA-MB-231 breast cancer cells and the normal breast epithelial cell line MCF10A. Testing in the isogenic BCA2 +ve MDA-MB-231/ER cell line restored antitumor activity for compounds that were inactive in the BCA2 -ve MDA-MB-231 cell line. In contrast, structurally related dithiocarbamates and benzisothiazolones (lacking the disulfide bond) were all inactive. Compounds 5d and 5f were additionally found to lack ALDH-inhibitory activity, suggestive of selective E3 ligase-inhibitory activity and worthy of further development.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/enzymology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Ubiquitin-Protein Ligases/antagonists & inhibitors , Aldehyde Dehydrogenase/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Disulfiram/chemical synthesis , Disulfiram/chemistry , Disulfiram/pharmacology , Disulfiram/therapeutic use , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/therapeutic use , Enzyme Stability , Gene Expression Regulation, Neoplastic/drug effects , Humans , Structure-Activity Relationship , Substrate Specificity , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/drug effects , Zinc/metabolismABSTRACT
Drug resistance remains a clinical challenge in cancer treatment due to poor understanding of underlying mechanisms. We have established several drug-resistant prostate cancer cell lines by long-term culture in medium containing chemotherapeutic drugs. These resistant lines displayed a significant increase in side population cells due to overexpression of drug efflux pumps including ABCG2/BCRP and MDR1/Pgp. To uncover potential mechanisms underlying drug resistance, we performed microarray analysis to identify differentially expressed genes in 2 drug-resistant lines. We observed that POU5F1/OCT4, a transcription factor key to regulating pluripotency in embryonic stem cells, was upregulated in drug-resistant lines and accompanied by transcriptional activation of a set of its known target genes. Upregulation of OCT4 in drug-resistant cells was validated by RT-PCR and sequencing of PCR products as well as confirmation by Western blot and specific shRNA knockdown. Analysis of the regulatory region of POU5F1/OCT4 revealed a reduction of methylation in drug-resistant cell lines. Furthermore, these drug-resistant cells exhibited a significant increase in tumorigenicity in vivo. Subcutaneous inoculation of as few as 10 drug-resistant cells could initiate tumor formation in SCID mice, whereas no detectable tumors were observed from the parental line under similar conditions, suggesting that these drug-resistant cells may be enriched for tumor-initiating cells. Knocking down OCT4 expression by specific shRNAs attenuated growth of drug-resistant cells. Our data suggest that OCT4 re-expression in cancer cells may play an important role in carcinogenesis and provide one possible mechanism by which cancer cells acquire/maintain a drug-resistant phenotype.
ABSTRACT
PURPOSE: Vorinostat, a histone deacetylase inhibitor, enhances cell death by the proteasome inhibitor bortezomib in vitro. We sought to test the combination clinically. EXPERIMENTAL DESIGN: A phase I trial evaluated sequential dose escalation of bortezomib at 1 to 1.3 mg/m2 i.v. on days 1, 4, 8, and 11 and vorinostat at 100 to 500 mg orally daily for 8 days of each 21-day cycle in relapsed/refractory multiple myeloma patients. Vorinostat pharmacokinetics and dynamics were assessed. RESULTS: Twenty-three patients were treated. Patients had received a median of 7 prior regimens (range, 3-13), including autologous transplantation in 20, thalidomide in all 23, lenalidomide in 17, and bortezomib in 19, 9 of whom were bortezomib-refractory. Two patients receiving 500 mg vorinostat had prolonged QT interval and fatigue as dose-limiting toxicities. The most common grade >3 toxicities were myelo-suppression (n = 13), fatigue (n = 11), and diarrhea (n = 5). There were no drug-related deaths. Overall response rate was 42%, including three partial responses among nine bortezomib refractory patients. Vorinostat pharmacokinetics were nonlinear. Serum Cmax reached a plateau above 400 mg. Pharmacodynamic changes in CD-138+ bone marrow cells before and on day 11 showed no correlation between protein levels of NF-kappaB, IkappaB, acetylated tubulin, and p21CIP1 and clinical response. CONCLUSIONS: The maximum tolerated dose of vorinostat in our study was 400 mg daily for 8 days every 21 days, with bortezomib administered at a dose of 1.3 mg/m2 on days 1, 4, 8, and 11. The promising antimyeloma activity of the regimen in refractory patients merits further evaluation.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Boronic Acids/administration & dosage , Hydroxamic Acids/administration & dosage , Multiple Myeloma/drug therapy , Pyrazines/administration & dosage , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Boronic Acids/adverse effects , Bortezomib , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Resistance, Neoplasm/drug effects , Female , Humans , Hydroxamic Acids/adverse effects , Hydroxamic Acids/pharmacokinetics , Injections, Intravenous , Male , Maximum Tolerated Dose , Middle Aged , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Pyrazines/adverse effects , Recurrence , VorinostatABSTRACT
PURPOSE: This study focuses on the synthesis and characterization of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-cyclo-RGD (Arg-Gly-Asp) conjugates for delivery of geldanamycin to prostate tumors. MATERIALS AND METHODS: HPMA copolymers containing aminohexylgeldanamycin (AH-GDM) with and without the targeting peptide RGDfK were synthesized and characterized. Drug release from copolymers was evaluated using cathepsin B. Competitive binding of copolymer conjugates to alpha(v)beta(3) integrin was evaluated in prostate cancer (PC-3) and endothelial (HUVEC) cell lines and in vitro growth inhibition was assessed. The maximum tolerated dose for single i.v. injections of free drug and the conjugates was established in nude mice. RESULTS: HPMA copolymers containing AH-GDM and RGDfK showed active binding to the alpha(v)beta(3) integrin similar to that of free peptide. Similarly, growth inhibition of cells by conjugates was comparable to that of the free drug. Single intravenous doses of HPMA copolymer-AH-GDM-RGDfK conjugates in mice were tolerated at 80 mg/kg drug equivalent, while free drug caused morbidity at 40 mg/kg. No signs of toxicity were present in mice receiving HPMA copolymer-AH-GDM-RGDfK over the 14-day evaluation period. CONCLUSION: Results of in vitro activity and in vivo tolerability experiments hold promise for the utility of HPMA copolymer-AH-GDM-RGDfK conjugates for treatment of prostate cancer with greater efficacy and reduced toxicity.
Subject(s)
Acrylamides/chemistry , Benzoquinones/administration & dosage , Drug Delivery Systems/methods , Lactams, Macrocyclic/administration & dosage , Peptides, Cyclic/chemistry , Prostatic Neoplasms/drug therapy , Acrylamides/metabolism , Acrylamides/toxicity , Animals , Benzoquinones/metabolism , Benzoquinones/toxicity , Cathepsin B/metabolism , Cell Line , Cell Proliferation/drug effects , Competitive Bidding , Female , Humans , Integrin alpha Chains/metabolism , Integrin beta Chains/metabolism , Lactams, Macrocyclic/metabolism , Lactams, Macrocyclic/toxicity , Male , Mice , Mice, Nude , Peptides, Cyclic/metabolism , Peptides, Cyclic/toxicity , Toxicity TestsABSTRACT
PURPOSE: This study aimed to identify and evaluate molecular targets for the development of a novel combination chemotherapy to treat refractory and recurrent diffuse large B-cell lymphoma (DLBCL). EXPERIMENTAL DESIGN: Lymphoma samples from 38 cases of primary and recurrent DLBCL were analyzed using real-time quantitative PCR of the RPS6KB1 and CDC2 genes, and immunohistochemistry for their gene products p70S6K/p85S6K and cdc2/cdk1. The Farage, Karpas422, Pfeiffer, and Toledo DLBCL cell lines were subsequently treated with rapamycin and UCN-01 alone or in combination. Cell proliferation, apoptosis, and cell cycle progression were analyzed after the drug treatment. In addition, the levels of several key protein kinases involved in the phosphoinositide 3'-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, apoptosis, and cell cycle progression were analyzed in the presence and absence of the drugs. RESULTS: Amplification of the RPS6KB1 and CDC2 genes was found in both primary and recurrent DLBCL. Moreover, the vast majority of these lymphomas (approximately 94%) were strongly positive for phospho-p70S6K and cdc2/cdk1 proteins. The combination of rapamycin and UCN-01 synergistically inhibited the DLBCL cell proliferation by inducing G1 arrest as well as apoptosis by suppressing the phosphorylation of p70S6K/p85S6K and CDC2 expression. CONCLUSION: RPS6KB1 and CDC2 overexpression is common in DLBCL. Simultaneously targeting the RPS6KB1 and CDC2 products phospho-p70S6K/p85S6K and cdc2/cdk1 is very effective in inhibiting DLBCL proliferation and overcoming drug resistance. This work suggests that multilevel inhibition of the PI3K/Akt/mTOR pathway and double-block of cell cycle progression are effective strategies for DLBCL therapy.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , CDC2 Protein Kinase/metabolism , Cyclin B/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Neoplasm Recurrence, Local/pathology , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Adult , Aged , Aged, 80 and over , Apoptosis/drug effects , Blotting, Western , CDC2 Protein Kinase/genetics , Cell Proliferation/drug effects , Cyclin B/genetics , Cyclin-Dependent Kinases , Drug Synergism , Female , Flow Cytometry , G1 Phase/drug effects , Gene Expression Profiling , Humans , Immunoenzyme Techniques , Lymphoma, Large B-Cell, Diffuse/metabolism , Male , Middle Aged , Neoplasm Recurrence, Local/metabolism , Oligonucleotide Array Sequence Analysis , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Protein Kinases/genetics , Protein Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribosomal Protein S6 Kinases, 70-kDa/genetics , Sirolimus/administration & dosage , Staurosporine/administration & dosage , Staurosporine/analogs & derivatives , TOR Serine-Threonine Kinases , Tumor Cells, CulturedABSTRACT
Sustained growth of solid tumours can rely on both the formation of new and the co-option of existing blood vessels. Current models suggest that binding of angiopoietin-2 (Ang-2) to its endothelial Tie2 receptor prevents receptor phosphorylation, destabilizes blood vessels, and promotes vascular permeability. In contrast, binding of angiopoietin-1 (Ang-1) induces Tie2 receptor activation and supports the formation of mature blood vessels covered by pericytes. Despite the intense research to decipher the role of angiopoietins during physiological neovascularization and tumour angiogenesis, a mechanistic understanding of angiopoietin function on vascular integrity and remodelling is still incomplete. We therefore assessed the vascular morphology of two mouse mammary carcinoma xenotransplants (M6378 and M6363) which differ in their natural angiopoietin expression. M6378 displayed Ang-1 in tumour cells but no Ang-2 in tumour endothelial cells in vivo. In contrast, M6363 tumours expressed Ang-2 in the tumour vasculature, whereas no Ang-1 expression was present in tumour cells. We stably transfected M6378 mouse mammary carcinoma cells with human Ang-1 or Ang-2 and investigated the consequences on the host vasculature, including ultrastructural morphology. Interestingly, M6378/Ang-2 and M6363 tumours displayed a similar vascular morphology, with intratumoural haemorrhage and non-functional and abnormal blood vessels. Pericyte loss was prominent in these tumours and was accompanied by increased endothelial cell apoptosis. Thus, overexpression of Ang-2 converted the vascular phenotype of M6378 tumours into a phenotype similar to M6363 tumours. Our results support the hypothesis that Ang-1/Tie2 signalling is essential for vessel stabilization and endothelial cell/pericyte interaction, and suggest that Ang-2 is able to induce a switch of vascular phenotypes within tumours.
Subject(s)
Angiopoietin-1/metabolism , Angiopoietin-2/pharmacology , Mammary Neoplasms, Experimental/blood supply , Neovascularization, Pathologic/pathology , Angiopoietin-1/analysis , Angiopoietin-2/metabolism , Animals , Cell Line, Tumor , Endothelial Cells/pathology , Mammary Neoplasms, Experimental/metabolism , Mice , Mice, Nude , Neoplasm Transplantation , Neovascularization, Pathologic/metabolism , Pericytes/pathology , Phenotype , Receptor, TIE-2/metabolism , Transplantation, HeterologousABSTRACT
Aberrant activation of the androgen receptor (AR) by the ErbB2/ErbB3 heterodimer contributes to the development of hormone resistance in prostate cancer. EBP1, an ErbB3-binding protein, acts as an AR corepressor. As EBP1 is decreased in preclinical models of hormone-refractory prostate cancer, we studied the expression of EBP1 in human prostate cancer. We found that the expression of the EBP1 gene was significantly decreased in prostate cancer tissues compared with benign prostate at both mRNA and protein levels. Restoration of EBP1 expression in the hormone-refractory LNCaP C81 cell line led to an amelioration of the androgen-independent phenotype based on established biological criteria and a reduction in the expression of a cohort of AR target genes. The ability of the ErbB3 ligand heregulin (HRG) to stimulate growth and AKT phosphorylation of hormone-refractory prostate cancer cells was abolished. Abrogation of EBP1 expression by short hairpin RNA in hormone-dependent LNCaP cells, which undergo apoptosis in response to HRG, resulted in HRG-stimulated cell growth. Restoration of EBP1 expression decreased the tumorigenicity of C81 xenografts in female mice, whereas elimination of EBP1 expression enhanced the ability of LNCaP cells to grow in female mice. Our data support a role for EBP1 in the development of hormone-refractory prostate cancer via inhibition of both AR- and HRG-stimulated growth and present a novel strategy for treating androgen-refractory prostate cancer.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Androgens/pharmacology , Drug Resistance, Neoplasm/drug effects , Prostatic Neoplasms/metabolism , RNA-Binding Proteins/metabolism , Receptor, ErbB-3/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Disease Progression , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Mice , Neuregulin-1/metabolism , Phenotype , Phosphatidylinositol 3-Kinases/metabolism , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/metabolism , RNA-Binding Proteins/genetics , Signal Transduction/drug effects , TransfectionABSTRACT
Inhibitors of the enzyme 17alpha-hydroxylase/17,20 lyase are a new class of anti-prostate cancer agents currently undergoing preclinical and clinical development. We have previously reported the superior anticancer activity of our novel 17alpha-hydroxylase/17,20 lyase inhibitor, VN/124-1, against androgen-dependent cancer models. Here, we examined the effect of VN/124-1 on the growth of the androgen-independent cell lines PC-3 and DU-145 and found that the compound inhibits their growth in a dose-dependent manner in vitro (GI50, 7.82 micromol/L and 7.55 micromol/L, respectively). We explored the mechanism of action of VN/124-1 in PC-3 cells through microarray analysis and found that VN/124-1 up-regulated genes involved in stress response and protein metabolism, as well as down-regulated genes involved in cell cycle progression. Follow-up real-time PCR and Western blot analyses revealed that VN/124-1 induces the endoplasmic reticulum stress response resulting in down-regulation of cyclin D1 protein expression and cyclin E2 mRNA. Cell cycle analysis confirmed G1-G0 phase arrest. Measurements of intracellular calcium levels ([Ca2+]i) showed that 20 micromol/L VN/124-1 caused a release of Ca2+ from endoplasmic reticulum stores resulting in a sustained increase in [Ca2+]i. Finally, cotreatment of PC-3 cells with 5, 10, and 20 micromol/L VN/124-1 with 10 nmol/L thapsigargin revealed a synergistic relationship between the compounds in inhibiting PC-3 cell growth. Taken together, these findings show VN/124-1 is endowed with multiple anticancer properties that may contribute to its utility as a prostate cancer therapeutic.
Subject(s)
Androstadienes/pharmacology , Benzimidazoles/pharmacology , Endoplasmic Reticulum/pathology , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/pathology , Steroid 17-alpha-Hydroxylase/antagonists & inhibitors , Androgens/metabolism , Androstadienes/chemistry , Animals , Benzimidazoles/chemistry , Calcium/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin D1/genetics , Down-Regulation/drug effects , Drug Synergism , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/genetics , Eukaryotic Initiation Factor-2/metabolism , G1 Phase/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Genes, Neoplasm , Male , Oligonucleotide Array Sequence Analysis , Phosphorylation/drug effects , Prostatic Neoplasms/genetics , Resting Phase, Cell Cycle/drug effects , Thapsigargin/pharmacology , Up-Regulation/drug effectsABSTRACT
An increased understanding of stem-cell biology at the molecular level, as well as the isolation and characterization of a rare subset of cells with tumor-initiating properties from several human tumor types, have renewed interest in the exploitation of cancer stem cells (CSCs) as therapeutic targets. Although CSCs share various characteristics with normal stem cells, including self-renewal, asymmetric cell division, indefinite proliferative capacity, and self-protection mechanisms, they also contain unique and disease-specific features suitable for exploitation as therapeutic targets. Several existing anticancer agents and experimental therapeutics can inhibit pathways critical to CSC maintenance, and could, therefore, be utilized to eradicate CSCs. In this review, general and tumor-type specific cancer-stem-cell targets are highlighted. In addition, known inhibitors of these targets that could be utilized in the design of clinical protocols together with conventional cytotoxics as debulking agents are described.
Subject(s)
Antineoplastic Agents/pharmacology , Neoplasms , Neoplastic Stem Cells , Biomarkers, Tumor/metabolism , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Humans , Membrane Transport Proteins/metabolism , Neoplasms/metabolism , Neoplasms/physiopathology , Neoplasms/therapy , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/physiologyABSTRACT
PURPOSE: KML001 (sodium metaarsenite) is an orally bioavailable arsenic compound that has entered phase I/II clinical trials in prostate cancer. In this study, we elucidated the mode of action of KML001 and investigated its effects on telomerase and telomeres. EXPERIMENTAL DESIGN: We compared telomere length to KML001 cytotoxic activity in a panel of human solid tumor cell lines. Duration of exposure and concentrations of KML001 that affect telomerase and telomeres were evaluated in relation to established mechanisms of arsenite action such as reactive oxygen species-related DNA damage induction. Binding of KML001 to telomeres was assessed by matrix-assisted laser desorption/ionization mass spectrometry. RESULTS: We established a significant inverse correlation (r(2) = 0.9) between telomere length and cytotoxicity. KML001 exhibited activity in tumor cells with short telomeres at concentrations that can be achieved in serum of patients. We found that telomerase is not directly inhibited by KML001. Instead, KML001 specifically binds to telomeric sequences at a ratio of one molecule per three TTAGGG repeats leading to translocation of the telomerase catalytic subunit into the cytoplasm. In prostate cancer cells with short telomeres, KML001 caused telomere-associated DNA damage signaling as shown by gamma-H2AX induction and chromatin immunoprecipitation assays as well as a rapid telomere erosion shown by metaphase fluorescence in situ hybridization. These effects were not seen in a lung cancer cell line with long telomeres. Importantly, arsenification of telomeres preceded DNA lesions caused by reactive oxygen species production. CONCLUSIONS: Sodium metaarsenite is a telomere targeting agent and should be explored for the treatment of tumors with short telomeres.
Subject(s)
Antineoplastic Agents/pharmacology , Arsenites/pharmacology , DNA Damage/drug effects , Prostatic Neoplasms/drug therapy , Sodium Compounds/pharmacology , Telomere/drug effects , Blotting, Western , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Cell Line, Tumor , Female , Fluorescent Antibody Technique , Humans , Immunoprecipitation , In Situ Hybridization , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Male , Prostatic Neoplasms/genetics , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Telomerase/drug effectsABSTRACT
This paper reports the synthesis of compounds formed by two indole systems separated by a heterocycle (pyridine or piperazine). As a primary screening, the new compounds were submitted to the National Cancer Institute for evaluation of antitumor activity in the human cell line screen. The pyridine derivatives were far more active than the piperazine derivatives. For the study of the mechanism of action, the most active compounds were subjected to COMPARE analysis and to further biological tests including proteasome inhibition and inhibition of plasma membrane electron transport. The compound bearing the 5-methoxy-2-indolinone moiety was subjected to the first in vivo experiment (hollow fiber assay) and was active. It was therefore selected for the second in vivo experiment (human tumor xenograft in mice). In conclusion we demonstrated that this approach was successful, since some of the compounds described are much more active than the numerous, so far prepared and tested 3-indolylmethylene-2-indolinones.