[Show abstract][Hide abstract] ABSTRACT: 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.
Evans Blue (10 mg/kg) and an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (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.
Evans Blue and FCE28068 displayed similar tumour levels in the range of 2-18 % 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.
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.
Cancer Chemotherapy and Pharmacology 06/2013; · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aminoflavone (AF) acts as a ligand of the aryl hydrocarbon receptor (AhR). Expression of estrogen receptor α (ERα) and AhR-mediated transcriptional induction of CYP1A1 can sensitize breast cancer cells to AF. The objective of this study was to investigate the combined antitumor effect of AF and the histone deacetylase inhibitor vorinostat for treating mesenchymal-like triple-negative breast cancer (TNBC) as well as the underlying mechanisms of such treatment.
In vitro antiproliferative activity of AFP464 (AF prodrug) in breast cancer cell lines was evaluated by MTS assay. In vitro, the combined effect of AFP464 and vorinostat on cell proliferation was assessed by the Chou-Talalay method. In vivo, antitumor activity of AFP464, given alone and in combination with vorinostat, was studied using TNBC xenograft models. Knockdown of ERα was performed using specific, small-interfering RNA. Western blot, quantitative RT-PCR, immunofluorescence, and immunohistochemical staining were performed to study the mechanisms underlying the combined antitumor effect.
Luminal and basal A subtype breast cancer cell lines were sensitive to AFP464, whereas basal B subtype or mesenchymal-like TNBC cells were resistant. Vorinostat sensitized mesenchymal-like TNBC MDA-MB-231 and Hs578T cells to AFP464. It also potentiated the antitumor activity of AFP464 in a xenograft model using MDA-MB-231 cells. In vitro and in vivo mechanistic studies suggested that vorinostat reactivated ERα expression and restored AhR-mediated transcriptional induction of CYP1A1.
The response of breast cancer cells to AF or AFP464 was associated with their gene expression profile. Vorinostat sensitized mesenchymal-like TNBC to AF, at least in part, by reactivating ERα expression and restoring the responsiveness of AhR to AF.
PLoS ONE 01/2013; 8(9):e74525. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BCA2 is an E3 ligase linked with hormone responsive breast cancers. We have demonstrated previously that the RING E3 ligase BCA2 has autoubiquitination activity and is a very unstable protein. Previously, only Rab7, tetherin, ubiquitin and UBC9 were known to directly interact with BCA2.
Here, additional BCA2 binding proteins were found using yeast two-hybrid and bacterial-II-hybrid screening techniques with Human breast and HeLa cDNA libraries. Co-expression of these proteins was analyzed through IHC of TMAs. Investigation of the molecular interactions and effects were examined through a series of in vivo and in vitro assays.
Ten unique BCA2 interacting proteins were identified, two of which were hHR23a and 14-3-3sigma. Both hHR23a and 14-3-3sigma are co-expressed with BCA2 in breast cancer cell lines and patient breast tumors (n = 105). hHR23a and BCA2 expression was significantly correlated (P = < 0.0001 and P = 0.0113) in both nucleus and cytoplasm. BCA2 expression showed a statistically significant correlation with tumor grade. High cytoplasmic hHR23a trended towards negative nodal status. Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2. Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a. On the other hand, phosphorylated BCA2 protein is stabilized by interaction with 14-3-3sigma both with and without proteasome inhibitor MG-132 suggesting that BCA2 is regulated by multiple degradation pathways.
The interaction between BCA2 and hHR23a in breast cancer cells stabilizes BCA2. High expression of BCA2 is correlated with grade in breast cancer, suggesting regulation of this E3 ligase is important to cancer progression.
[Show abstract][Hide abstract] ABSTRACT: Mammalian target of rapamycin (mTOR) inhibitors mediate AKT activation through a type 1 insulin-like growth factor receptor (IGF-1R)-dependent mechanism. Combining the mTOR inhibitor temsirolimus with cixutumumab, a fully human immunoglobulin G1 monoclonal antibody directed against IGF-1R, was expected to enhance mTOR-targeted anticancer activity by modulating resistance to mTOR inhibition. The objectives of this phase I study were to evaluate the tolerability and activity of temsirolimus and cixutumumab.
Patients in sequential cohorts ("3 + 3" design) received escalating doses of temsirolimus with cixutumumab weekly for 28 days. At the maximum tolerated dose (MTD), 21 patients were randomized into three separate drug sequence treatment groups for serial blood draws and 2[18F]fluoro-2-deoxy-d-glucose positron emission tomography combined with X-ray computed tomography (FDG-PET/CT) scans for pharmacodynamic analyses (PD).
Forty-two patients with advanced cancer (19 male/23 female, median age = 53, median number of prior therapies = 4) were enrolled. MTD was reached at cixutumumab, 6 mg/kg IV and temsirolimus, 25 mg IV. Dose-limiting toxicities included grade 3 mucositis, febrile neutropenia, and grade 4 thrombocytopenia. The most frequent toxicities were hypercholesterolemia, hypertriglyceridemia, hyperglycemia, thrombocytopenia, and mucositis. Tumor reduction was observed in 2 of 3 patients with Ewing's sarcoma and in 4 of 10 patients with adrenocortical carcinoma. PD data suggest that cixutumumab alone or combined with temsirolimus increased plasma IGF-1 and IGF binding protein 3. FDG-PET/CT showed the odds of achieving stable disease decreased by 58% (P = 0.1213) with a one-unit increase in absolute change of standard uptake value from baseline to day 3.
Temsirolimus combined with cixutumumab was well tolerated. We are currently enrolling expansion cohorts at the MTD for Ewing's sarcoma and adrenocortical carcinoma.
Clinical Cancer Research 07/2011; 17(18):6052-60. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Breast ultrasound tomography is a rapidly developing imaging modality that has the potential to impact breast cancer screening and diagnosis. Double difference (DD) tomography utilizes more accurate differential time-of-flight (ToF) data to reconstruct the sound speed structure of the breast. It can produce more precise and better resolution sound speed images than standard tomography that uses absolute ToF data. We apply DD tomography to phantom data and excised mouse mammary glands data. DD tomograms demonstrate sharper sound speed contrast than the standard tomograms.
[Show abstract][Hide abstract] ABSTRACT: Disulfiram is a FDA approved drug for the treatment of alcoholism and available for clinical use since over 5 decades. Despite data from the 1970s and 80s that showed that disulfiram and analogs are able to enhance the activity of anticancer cytotoxic drugs and might be useful chemopreventative agents, the underlying molecular mechanisms remained unknown until recently. Large scale screening efforts for agents that can inhibit the proteasome and be used as novel anticancer drugs, revealed that disulfiram has proteasome inhibitory activity. Moreover, disulfiram was also found to have specific activity against zinc fingers and RING-finger ubiquitin E3 ligases that play an important role in cancer development. Here, we review the preclinical and clinical studies exploring disulfiram as an anticancer agent as well as research programs that focus on the development of disulfiram derivatives as inhibitors of the ubiquitin-proteasome system.
Current cancer drug targets 03/2011; 11(3):338-46. · 5.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 03/2011; 6(5):927-33. · 4.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since the first report of the successful xenografting of a human tumor into nude mice in 1969, there have been numerous studies
conducted throughout the world using the nude moue as a tool to answer a variety of questions regarding the cause, prevention,
and therapy of cancer. Thus, the role of immunodeficient animals in oncology has continuously increased and the athymic nude
mouse has proven to be an outstanding host for many human solid tumor xenografts [1, 2]. The latter are now extensively used
in the development of potential anticancer drugs, new antineoplastic treatment modalities, and studies on tumor biology [3–7].
Moreover, mice with severe combined immunodeficiency (SCID) and nonobese diabetic (NOD)/SCID mice have enlarged the spectrum
of possible applications in cancer research. SCID mice have enabled engraftments of human tumors which were previously difficult
to explant such as those of the hematopoietic system . The NOD/SCID mouse tumor repopulation model has become the gold
standard for the analysis of self-renewal properties of putative cancer stem cells and is critical to the emerging field of
cancer stem cell biology .
KeywordsHuman Xenografts-Patient derived tumor models-Molecular characterization-Drug sensitivity testing-Gene signature
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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
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.
Clinical Cancer Research 07/2010; 16(14):3795-804. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Medicinal Chemistry 03/2010; 53(7):2757-65. · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The expression of side-population (SP) cells and their relation to tumour-initiating cells (T-ICs) have been insufficiently studied in breast cancer (BC). We therefore evaluated primary cell cultures derived from patients and a panel of human BC cell lines with luminal- or basal-molecular signatures for the presence of SP and BC stem cell markers.
The SPs from luminal-type BC were analysed for BC T-IC characteristics, including human epidermal growth factor receptor 2 (HER2), ERalpha, IGFBP7 expression and their ability to initiate tumours in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice. Pharmacological modulators were used to assess the effects of HER2 signalling and breast cancer-resistance protein (BCRP) expression on SPs.
The SP was more prevalent in the luminal subtype of BC compared with the basal subtype. HER2 expression was significantly correlated with the occurrence of an SP (r(2)=0.75, P=0.0003). Disappearance of SP in the presence of Ko143, a specific inhibitor of the ATP-binding cassette transporter BCRP, suggests that BCRP is the predominant transporter expressed in this population. The SP also decreased in the presence of HER2 signalling inhibitors AG825 or trastuzumab, strengthening the notion that HER2 contributed to the SP phenotype, likely through downstream AKT signalling. The SP cells from luminal-type MCF-7 cells with enforced expression of HER2, and primary cells with luminal-like properties from a BC patient, displayed enrichment in cells capable of repopulating tumours in NOD/SCID mice. Engraftment of SP cells was inhibited by pretreatment with AG825 or by in vivo treatment with trastuzumab.
Our findings indicate an important role of HER2 in regulating SP and hence T-ICs in BC, which may account for the poor responsiveness of HER2-positive BCs to chemotherapy, as well as their aggressiveness.
British Journal of Cancer 02/2010; 102(5):815-26. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Letters in Drug Design & Discovery 01/2010; 7(1):57-62. · 0.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Telomeres and telomerase are targets for anticancer drug development and specific inhibitors are currently under clinical investigation. However, it has been reported that standard cytotoxic agents can affect telomere length and telomerase activity suggesting that they also have of a role in drug resistance. in this study, telomere lengths and telomerase activity as well as drug efflux pump expression, glutathione (GSH) levels and polyadenosine-ribose polymerase (PARP) cleavage were assessed in a panel of human tumor cell lines made resistant to vindesine, gemcitabine and cisplatin. these included two lung cancer cell lines resistant to vindesine (LXFL 529L/Vind, LXFA 526L/Vind), a renal cancer cell line (RXF944L/Gem) and an ovarian cancer cell line (AG6000) resistant to gemcitabine, and one resistant to cisplatin (ADDP). The resistant clones were compared to their parental lines and evaluated for cross resistance to other cytotoxic agents. Several drug specific resistance patterns were found, and various complex patterns of cross resistance emerged from some cell lines, but these mechanisms of resistance could not be related to drug efflux pump expression, GSH levels or pARp cleavage. However, all displayed changes in telomerase activity and/or telomere length. Our studies present evidence that telomere maintenance should be taken into consideration in efforts not only to overcome drug resistance, but also to optimize the use of telomere-based therapeutics.
[Show abstract][Hide abstract] ABSTRACT: The present invention relates to a method of treating cancer in a subject in need thereof, by administering to the subject a combination of sodium meta arsenite and/or arsenic trioxide and a cytotoxic cancer agent, such as cisplatin, adriamycin, and taxane, such as larotaxel, orataxel, tesetaxel, docetaxel or paclitaxel. The arsenic compound(s) and cytotoxic anti-cancer agent may be administered together or in a composition or separately as a combination therapy
Ref. No: USA Patent Application 20090246291, Year: 10/2009
[Show abstract][Hide abstract] ABSTRACT: Vorinostat, a histone deacetylase inhibitor, enhances cell death by the proteasome inhibitor bortezomib in vitro. We sought to test the combination clinically.
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.
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.
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.
Clinical Cancer Research 09/2009; 15(16):5250-7. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ubiquitin E3 ligases are the most prevalent cancer genes besides protein kinases, suggesting a critical role in cancer development
and progression. Two major classes of E3 ligases can be distinguished: the HECT-type and the adaptor-type E3s. The latter
include RING-finger, PHD, and U-box domain ligases. The vast majority of the known and cancer-associated E3s belong to the
RING-finger gene family. This chapter describes key representatives of each class of E3s, their substrates, and relevance
KeywordsUbiquitin ligase–Ubiquitin–RING-type E3 ligase–HECT-type E3 ligase–Cancer