ABSTRACT
BACKGROUND: Administration of certain chemotherapy drugs at the maximum tolerated dose, vascular-disrupting agents (VDAs) and irradiation can induce mobilisation and tumour homing of proangiogenic bone marrow-derived cells (BMDCs). Increases in cytokines and chemokines contribute to such mobilisation that eventually promotes tumour (re)growth. NGR-TNF is a vascular-targeting agent in advanced clinical development, coupling the CNGRCG angiogenic vessel-homing peptide with tumour necrosis factor-alpha (TNF). We investigated whether NGR-TNF mobilises host BMDCs and growth factors. METHODS: Blood was obtained from Lewis lung carcinoma and 4T1 tumour-bearing mice at different time points following NGR-TNF, VDA or anti-VEGFR2/flk-1 antibody treatment. Levels of circulating growth factors were assessed by ELISAs. BMDCs were characterised by FACS. Circulating endothelial progenitor cells were defined as CD45(-)/CD13(+)/flk-1(+)/CD117(+)/7AAD(-), Tie2-expressing monocytes as CD45(+)/CD11b(+)/Tie2(+) and myeloid-derived suppressor cells as CD45(+)/CD11b(+)/Gr1(+) cells. RESULTS: NGR-TNF decreases tumour blood vessel density-inducing apoptosis of tumour and tumour endothelial cells. Unlike VDAs, or high-dose NGR-TNF, lower doses of NGR-TNF, comparable to those used in clinical trials, neither mobilise nor recruit to the tumour site proangiogenic BMDCs or induce host growth factors. CONCLUSION: Low-dose NGR-TNF exerts antitumour activity without inducing proangiogenic host responses, conceivably important for preventing/overcoming resistance and designing combination therapeutic strategies.
Subject(s)
Angiogenesis Inhibitors/administration & dosage , Bone Marrow Cells/drug effects , Chemotaxis/drug effects , Cytokines/pharmacology , Molecular Targeted Therapy , Tumor Necrosis Factor-alpha/administration & dosage , Animals , Apoptosis/drug effects , Blood Vessels/drug effects , Bone Marrow Cells/pathology , Bone Marrow Cells/physiology , Carcinoma, Lewis Lung/drug therapy , Carcinoma, Lewis Lung/pathology , Cell Line, Tumor , Cytokines/metabolism , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Female , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neovascularization, Pathologic/pathologyABSTRACT
Metronomic chemotherapy refers to the administration of chemotherapy at low, nontoxic doses on a frequent schedule with no prolonged breaks. The aim of the study is to rationally develop a CPT-11 metronomic regimen in preclinical settings of colon cancer. In vitro cell proliferation, apoptosis and thrombospondin-1/vascular endothelial growth factor (TSP-1/VEGF) expression analyses were performed on endothelial (HUVEC, HMVEC-d) and colorectal cancer (HT-29, SW620) cells exposed for 144 h to metronomic concentrations of SN-38, the active metabolite of CPT-11. HT-29 human colorectal cancer xenograft model was used, and tumour growth, microvessel density and VEGF/TSP-1 quantification was performed in tumours. In vitro and in vivo combination studies with the tyrosine inhibitor semaxinib were also performed. SN-38 preferentially inhibited endothelial cell proliferation alone and interacted synergistically with semaxinib; it induced apoptosis and increased the expression and secretion of TSP-1. Metronomic CPT-11 alone and combined with semaxinib significantly inhibits tumour growth in the absence of toxicity, which was accompanied by decreases in microvessel density and increases in TSP-1 gene expression in tumour tissues. In vitro results show the antiangiogenic properties of low-concentration SN-38, suggesting a key role of TSP-1 in this effect. In vivo, the CPT-11 metronomic schedule is effective against tumour and microvessel growth without toxic effect on mice.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Camptothecin/analogs & derivatives , Colorectal Neoplasms/blood supply , Colorectal Neoplasms/drug therapy , Indoles/pharmacology , Pyrroles/pharmacology , Angiogenesis Inhibitors/administration & dosage , Animals , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Apoptosis/drug effects , Camptothecin/administration & dosage , Camptothecin/pharmacology , Cell Proliferation/drug effects , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation, Neoplastic/drug effects , HT29 Cells , Humans , Immunoenzyme Techniques , Immunohistochemistry , Indoles/administration & dosage , Irinotecan , Male , Mice , Mice, Nude , Microcirculation/drug effects , Pyrroles/administration & dosage , Thrombospondin 1/drug effects , Thrombospondin 1/metabolism , Transplantation, Heterologous , Vascular Endothelial Growth Factor A/drug effects , Vascular Endothelial Growth Factor A/metabolismABSTRACT
The pharmacokinetics (PK) and pharmacodynamics (PD) of metronomic irinotecan have not been studied in cancer patients. The aim of the study is to investigate the PK/PD profile of irinotecan/SN-38 administered by metronomic schedule. Twenty chemotherapy-refractory or chemotherapy-resistant patients with metastatic colorectal carcinoma were enrolled. Irinotecan was infused continuously as follows: irinotecan 1.4 mg m(-2) day(-1) (n=7), 2.8 mg m(-2) day(-1) (n=5) and 4.2 mg m(-2) day(-1) (n=8). Drug levels were examined by HPLC, whereas ELISAs and real-time RT-PCR were used, respectively, for the measurement of plasma levels and gene expression in peripheral blood mononuclear cells of vascular endothelial growth factor/thrombospondin-1. Pharmacokinetic analysis demonstrated that the steady-state levels (C(ss)) of SN-38 were between 1 and 3.3 ng ml(-1). From a PD point of view, higher thrombospondin-1 (TSP-1) plasma levels (153.4+/-30.1 and 130.4+/-9.2% at day 49 vs pretreatment values at 1.4 and 2.8 mg m(-2) day(-1) dose levels, respectively) and increased gene expression in PBMC were found during the metronomic irinotecan infusion, especially at the lower doses. Four patients (20%) obtained a stable disease (median 3.9 months) despite progressing during previous standard irinotecan schedule. Toxicities >grade 1 were not observed. Metronomic irinotecan administration is very well tolerated and induces an increase of gene expression and plasma concentration of TSP-1 at low plasma SN-38 concentrations.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacokinetics , Camptothecin/analogs & derivatives , Colorectal Neoplasms/drug therapy , Aged , Camptothecin/administration & dosage , Camptothecin/adverse effects , Camptothecin/pharmacokinetics , Camptothecin/pharmacology , Colorectal Neoplasms/mortality , Female , Granulocyte Colony-Stimulating Factor/pharmacology , Humans , Irinotecan , Male , Middle Aged , Thrombospondin 1/blood , Thrombospondin 1/genetics , Vascular Endothelial Growth Factor A/blood , Vascular Endothelial Growth Factor A/geneticsABSTRACT
BACKGROUND: Metronomic chemotherapy-the chronic administration of chemotherapy at relatively low, minimally toxic doses on a frequent schedule of administration at close regular intervals, with no prolonged drug-free breaks-is a potentially novel approach to the control of advanced cancer disease. It is thought to work primarily through antiangiogenic mechanisms and has, as an advantage, the property of significantly reducing undesirable toxic side-effects. The aim of the present study was to evaluate the cost effectiveness of cyclophosphamide-methotrexate 'metronomic' chemotherapy in the palliative treatment of pretreated metastatic breast cancer. METHODS: Low-dose cyclophosphamide-methotrexate 'metronomic' chemotherapy was compared with outcome and resource utilisation data of published phase II trials regarding metastatic breast cancer, performed in western countries, mostly in Europe. All direct costs associated with metastatic breast cancer treatment were included and adjusted to year 2003 values. Sensitivity analyses were performed and variations to the values of key parameters were assessed. RESULTS: Low-dose cyclophosphamide-methotrexate 'metronomic' therapy was assessed to be a cost-effective/cost-saving therapy for palliative treatment for metastatic breast cancer when compared with novel chemotherapy strategies (phase II trials). Compared with the 11 phase II mono- and combination chemotherapies, metronomic treatment showed marked cost savings in each case and improved cost effectiveness. Sensitivity analyses showed the results were robust to variations to the values of key parameters with very few exceptions. CONCLUSIONS: Metronomic cyclophosphamide-methotrexate is significantly cost effective. If validated by prospective randomized trials, the treatment concept could reduce healthcare costs, especially those associated with the combined use of new, highly expensive, molecularly targeted therapies.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/economics , Breast Neoplasms/drug therapy , Breast Neoplasms/economics , Administration, Oral , Breast Neoplasms/secondary , Clinical Trials, Phase II as Topic , Cost-Benefit Analysis , Cyclophosphamide/administration & dosage , Cyclophosphamide/economics , Drug Costs , Economics, Pharmaceutical , Humans , Infusions, Intravenous , Methotrexate/administration & dosage , Methotrexate/economics , Palliative CareABSTRACT
Intrinsic alterations in the tumor microenvironment are known to contribute to various forms of drug resistance. For example, tumor hypoxia, due to abnormal or sluggish blood flow within areas of solid tumors, can result in both microenvironment-mediated radiation and chemotherapeutic drug resistance. In contrast, acquired resistance to chemotherapy is generally considered to be the result of the gradual selection of mutant subpopulations having genetic mutations and biochemical alterations responsible for the resistant phenotype. Here we present a paradigm for therapyinduced microenvironment-mediated acquired drug resistance. It is based on the results showing that tumor cells appear to be heterogeneous in their relative dependence on adjacent tumor-associated vasculature for survival. Some tumor cells are highly vessel dependent, whereas some are significantly less so, and thus can survive in more hypoxic regions of tumors, distal from such tumor vessels. Hence, it is possible that variant tumor cells that are less vessel dependent may therefore be selected for over time by successful antiangiogenic drug therapies. This results in loss of response or attenuated responses to the therapy. Preliminary evidence is summarized in support of this hypothesis, using paired human colon cancer (HCT116) cell lines that contain two copies of either the wild-type or the disrupted p53 tumor suppressor gene. The mutant cells were found to be less responsive to antiangiogenic therapy, compared to the wild-type cells, and could be progressively selected for in mixed cell populations. Because p53 inactivation can lead to resistance to hypoxia-mediated apoptosis, the results suggest that a protracted and successful antiangiogenic therapy may create more hypoxic tumor microenvironments, thereby creating the necessary conditions to accelerate the selection of mutant tumor cells that are more adept in surviving and growing in such environments. As such, consideration might be given to the combined use of bioreductive hypoxic cell cytotoxic drugs and angiogenesis inhibitors to prolong the efficacy of antiangiogenic therapeutics.
Subject(s)
Drug Resistance, Neoplasm , Neoplasms/drug therapy , Animals , Apoptosis/physiology , Cell Communication , Humans , Hypoxia/metabolism , Mice , Neoplasms/pathology , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismSubject(s)
Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Neoplasms/drug therapy , Neovascularization, Pathologic/drug therapy , Clinical Trials as Topic , Dose-Response Relationship, Drug , Endothelial Growth Factors/metabolism , Female , Humans , Lymphokines/metabolism , Neoplasms/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Tek/Tie-2 is an endothelial cell (EC)-specific receptor tyrosine kinase that plays a critical role in angiogenesis via its regulation by the angiopoietin family of growth factor ligands. Angiopoietin-1 (Ang1) can promote EC migration; however, the signaling mechanisms underlying this process remain elusive. Here we demonstrate that Dok-R/Dok-2 can associate with Tek in ECs following Ang1 stimulation, resulting in tyrosine phosphorylation of Dok-R and the subsequent recruitment of Nck and the p21-activating kinase (Pak/Pak1) to the activated receptor. Ang1-mediated migration is increased upon Dok-R overexpression and this requires a functional Nck binding site on Dok-R. Localization of this Dok-R-Nck-Pak complex to the activated Tek receptor at the cellular membrane is coincident with activation of Pak kinase. The ability of Dok-R to bind Nck is required for maximal activation of Pak and overexpression of Pak results in increased Ang1-mediated cell motility. Our study outlines a novel signaling pathway underlying Ang1-driven cell migration that involves Dok-R and its recruitment of Nck and the subsequent activation of Pak.
Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/metabolism , Membrane Glycoproteins/pharmacology , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Angiopoietin-1 , Binding Sites/physiology , Carrier Proteins/genetics , Cell Line , Cell Movement/drug effects , Cell Movement/physiology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Enzyme Activation/drug effects , Enzyme Activation/physiology , Epithelial Cells/cytology , Epithelial Cells/metabolism , Humans , Kidney/cytology , Kidney/drug effects , Kidney/metabolism , Macromolecular Substances , Oncogene Proteins/metabolism , Phosphoproteins/genetics , Phosphorylation/drug effects , Protein Binding/physiology , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, TIE-2 , Signal Transduction/drug effects , Signal Transduction/physiology , p21-Activated KinasesABSTRACT
BACKGROUND & AIMS: Advanced gastric cancer has a poor prognosis and is largely unresponsive to currently available chemotherapeutic drugs. The development of more effective therapies would be aided by better preclinical models. METHODS: An in vitro multicellular gastric cancer spheroid model was established using the liquid overlay technique and compared with the corresponding xenografts in immunodeficient mice. RESULTS: Twelve of 17 (71%) gastric cancer cell lines reflected growth characteristics of their parental gastric carcinomas in three-dimensional culture. Thus, cell lines derived from peritoneal and pleural carcinomatosis grew as single cells (HSC-39, KATO-II, KATO-III) and cell aggregates (SNU-5, SNU-16). Cell lines representing adenosquamous (MKN-1) and tubular differentiation (MKN-28, MKN-74, N87) formed partly compact multicellular spheroids recapitulating the tumor architecture of the respective original tumor. The differentiated phenotype was lost after subcutaneous implantation of the in vitro spheroids in mice. The degree of morphologic differentiation was reflected by the levels of mucin and constitutive E-cadherin expression. Heterogeneous changes of other adhesion molecules (EpCAM, alpha2beta1, CD44s, Le(x), sLe(x)) were observed. In contrast, cell lines derived from poorly differentiated gastric carcinomas (Hs-746T, RF-1, RF-48) formed fully compact spheroids mimicking the poorly differentiated phenotype, were E-cadherin negative, and showed only CD44s up-regulation. CONCLUSIONS: Recapitulating some complexity of their in vivo counterparts, multicellular gastric cancer spheroids may represent a physiologically valid model for studying the biology of this cancer, and testing new therapeutic strategies.
Subject(s)
Spheroids, Cellular/cytology , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Animals , Cell Aggregation , Cell Differentiation , Cell Division , Female , Humans , Mice , Mice, Nude , Phenotype , Stomach Neoplasms/classification , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
Normal or immortal epithelial cells are sensitive to a form of apoptosis, commonly referred to as anoikis, which is induced by detachment from the extracellular matrix (ECM). In contrast, development of carcinomas is associated with acquisition of cellular resistance to anoikis. However, whether human cancer cells deprived of anoikis resistance necessarily display reduced tumorigenic properties in vivo is unknown. We decided to address this question using human ovarian carcinoma cells as a model. Bcl-X(L), an apoptotic factor considered to play an important role in (resistance to) anoikis, is overexpressed in ovarian cancer, and represents an unfavorable prognostic indicator for this type of human malignancy. We therefore evaluated whether Bcl-X(L) can be used as a tool to manipulate anoikis resistance and tumorigenicity of ovarian cancer cells. We show here that when nonmalignant ovarian epithelial cells are detached from the ECM, down-regulation of Bcl-X(L) and apoptotic cell death are observed, although these events do not occur in ovarian carcinoma cells. Moreover, enforced down-regulation of Bcl-X(L) by transfection with antisense cDNA in the anoikis-resistant and highly tumorigenic HEY ovarian carcinoma cell line had no impact on the viability of these cells under adherent conditions but caused significant apoptosis in response to detachment from the ECM. This change was associated with a strong inhibition of tumorigenicity of the Bcl-X(L)-deficient HEY cells in nude mice, both s.c. and in the peritoneal cavity. These results suggest a critical role for Bcl-X(L) in the maintenance of anoikis resistance in ovarian cancer cells. They also serve to establish a functional linkage between this property and the ability of human cancer cells to grow aggressively in vivo. Consequently, targeting molecular mechanisms responsible for anoikis resistance may serve as a potentially effective therapeutic strategy for the treatment of such human malignancies as ovarian cancer.
Subject(s)
Anoikis/physiology , Ovarian Neoplasms/pathology , Proto-Oncogene Proteins c-bcl-2/physiology , Cell Division/physiology , DNA, Antisense/genetics , DNA, Complementary/genetics , Down-Regulation , Epithelial Cells/cytology , Epithelial Cells/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins c-bcl-2/genetics , Spheroids, Cellular/pathology , Transfection , bcl-X ProteinABSTRACT
Inhibitors of epidermal growth factor receptor (EGFR) signaling are among the novel drugs showing great promise for cancer treatment in the clinic. However, the possibility of acquired resistance to such drugs because of tumor cell genetic instabilities has not yet been explored. Here we report the experimental derivation and properties of such cell variants obtained from recurrent tumor xenografts of the human A431 squamous cell carcinoma, after two consecutive cycles of therapy with one of three different anti-EGFR monoclonal antibodies: mR3, hR3, or C225. Initial response to a 2-week period of treatment was generally total tumor regression and was not significantly different among the three antibody groups. However, tumors often reappeared at the site of inoculation, generally after prolonged latency periods, and most of the tumors became refractory to a second round of therapy. Cell lines established from such resistant tumors retained high EGFR expression, normal sensitivity to anti-EGFR antibody or ligand, and unaltered growth rate when compared with the parental line in vitro. In contrast, the A431 cell variants exhibited an accelerated growth rate and a significantly attenuated response to anti-EGFR antibodies in vivo relative to the parental line. Because of the reported suppressive effect of EGFR inhibitors on vascular endothelial growth factor (VEGF) expression, and the demonstrated role of VEGF in the angiogenesis and growth of A431 tumor xenografts, relative VEGF expression was examined. Five of six resistant variants expressed increased levels of VEGF, which paralleled an increase in both angiogenic potential in vitro and tumor angiogenesis in vivo. In addition, elevated expression of VEGF in variants of A431 cells obtained by gene transfection rendered the cells significantly resistant to anti-EGFR antibodies in vivo. Taken together, the results suggest that, at least in the A431 system, variants displaying acquired resistance to anti-EGFR antibodies can emerge in vivo and can do so, at least in part, by mechanisms involving the selection of tumor cell subpopulations with increased angiogenic potential.
Subject(s)
Antibodies, Monoclonal/pharmacology , Carcinoma, Squamous Cell/blood supply , ErbB Receptors/antagonists & inhibitors , Neovascularization, Pathologic/pathology , Animals , Antibodies, Monoclonal/immunology , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/therapy , Drug Resistance, Neoplasm , Endothelial Growth Factors/biosynthesis , Endothelial Growth Factors/genetics , Endothelial Growth Factors/physiology , ErbB Receptors/immunology , Humans , Lymphokines/biosynthesis , Lymphokines/genetics , Lymphokines/physiology , Mice , Mice, SCID , Neoplasm Recurrence, Local , Neovascularization, Pathologic/metabolism , Tumor Cells, Cultured , Up-Regulation , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Xenograft Model Antitumor AssaysABSTRACT
Given the multifaceted role of Ras in tumor angiogenesis, pharmacologic targeting of such proteins may bring about at least three important consequences: (1) partial obliteration of the angiogenic competence of tumor cells, (2) an increase in vascular dependence and sensitization to apoptosis, and (3) a direct inhibition of endothelial cell responses to proangiogenic stimuli. Exploration of some of these possibilities, using various pharmacological compounds and antibodies, has already begun. An intriguing possibility is that Ras antagonists and signal transduction inhibitors may synergize with a number of other antiangiogenic modalities such as direct acting antiangiogenic agents (e.g., endostatin) or antivascular regimens involving low-dose continuous chemotherapy as a vasculature-targeting strategy.
Subject(s)
Endothelial Growth Factors/genetics , Lymphokines/genetics , Neoplasms/blood supply , Neovascularization, Pathologic/genetics , ras Proteins/genetics , 3T3 Cells , Animals , Cell Line , Cell Transformation, Neoplastic , Endothelial Growth Factors/metabolism , Humans , Lymphokines/metabolism , Mice , Mutation , Neovascularization, Pathologic/metabolism , Up-Regulation , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , ras Proteins/metabolismABSTRACT
Cells within a tumor are highly heterogeneous with respect to a wide range of genotypic and phenotypic characteristics. The latter include such properties as growth, survival, invasion, and metastasis. We asked whether the degree to which individual tumor cells rely on a tumor's vasculature might also be heterogeneous. By adapting an intravital Hoechst 33342 staining technique, we labeled and isolated tumor cells based on their relative proximity to perfused vessels. Because tumor regions distal to the vasculature are likely hypoxic, we examined cells deficient for hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor that has been shown to mediate hypoxia-induced responses, including apoptosis. Despite reduced vascularization in HIF-1alpha-/- embryonic stem cell-derived tumors, their growth in vivo was found to be accelerated relative to HIF-1alpha+/+ tumor counterparts. We hypothesized that this paradoxical observation is because of decreased apoptotic rate, resulting in diminished vascular dependence of HIF-1alpha-/- cells. Analysis of heterogeneous tumors established from mixtures of HIF-1alpha+/+ with HIF-1alpha-/- cells revealed that the proportion of cells expressing wild-type HIF-1alpha was increased in perivascular areas and decreased in distal tumor regions. Thus, cells expressing HIF-1alpha were found to be highly dependent on proximity to blood vessels for their growth and survival in vivo, whereas cells that had lost HIF-1alpha expression were much less so. Heterogeneity in angiogenesis dependence was also observed among cell subpopulations isolated from human melanoma xenografts. This potential for selection of less vascular-dependent tumor cell variants throughout the course of disease progression may have important implications for the long-term efficacy of anti-angiogenic therapy.
Subject(s)
Neoplasms/blood supply , Neoplasms/physiopathology , Transcription Factors , Animals , Benzimidazoles , Blood Vessels/physiopathology , Cell Division , DNA-Binding Proteins/metabolism , Fluorescent Dyes , Genotype , Humans , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Melanoma/blood supply , Melanoma/pathology , Melanoma/physiopathology , Mice , Mice, Nude , Neoplasm Transplantation , Neoplasms/pathology , Nuclear Proteins/metabolism , Teratoma/blood supply , Teratoma/pathology , Teratoma/physiopathology , Transplantation, HeterologousABSTRACT
A major obstacle in the clinical management of malignant melanoma is its intrinsic resistance to chemotherapy and radiation therapy. Consequently, most patients with melanoma often do not respond to conventional anticancer therapy in a clinically significant manner. Recent advances in cancer research have provided new insights into the mechanisms of intrinsic resistance in melanomas. We have recently reported that the over-expression of tyrosinase-related protein 2 (TYRP2), an enzyme that is well characterized for its function in melanin synthesis, is associated specifically with resistance to DNA damaging drugs and radiation treatment. This review will summarize our findings as well as discuss the possible mechanisms by which TYRP2 over-expression contributes to intrinsic resistance in human malignant melanoma.
Subject(s)
Antineoplastic Agents/therapeutic use , Cell Lineage/physiology , Drug Resistance, Neoplasm , Intramolecular Oxidoreductases/metabolism , Melanoma/enzymology , Radiation Tolerance , Humans , Melanoma/drug therapy , Melanoma/radiotherapyABSTRACT
The ultimate target of anti-angiogenic drugs is the genetically stable, activated endothelial cell of a newly forming tumor blood vessel, rather than the genetically unstable tumor cell population per se. This led to the notion that acquired resistance to such drugs may not develop as readily, if at all. While there is some evidence that this lack of resistance development may be the case for some direct-acting angiogenesis inhibitors, it is becoming apparent that resistance can develop over time to many types of angiogenesis inhibitors including, possibly, some direct inhibitors, especially when used as monotherapies. Possible mechanisms for such acquired or induced resistance include: (i) redundancy of pro-angiogenic growth factors when the drug used targets a single such growth factor or its cognate endothelial cell-associated receptor tyrosine kinase; (ii) the anti-apoptotic/pro-survival function of growth factors such as VEGF, which, in high local concentrations, can antagonize the pro-apoptotic effects of various angiogenesis inhibitors; (iii) epigenetic, transient upregulation, or induction, of various anti-apoptotic effector molecules in host-endothelial cells; and (iv) heterogeneous vascular dependence of tumor cell populations. It is suggested that long-term disease control with anti-angiogenic drugs can be best achieved by judicious combination therapy. In this regard, the great molecular diversity of anti-angiogenic drug targets, in contrast to chemotherapy, makes this a particularly attractive therapeutic option, especially when approved, commercially available drugs considered to have anti-angiogenic effects are used in such combination treatment strategies.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Drug Resistance, Neoplasm , Animals , Drug Therapy, Combination , Humans , Neoplasms/blood supply , Neoplasms/drug therapyABSTRACT
Tyrosinase-related protein-2 (TYRP2) is a melanocyte-specific enzyme that catalyses the non-decarboxylative tautomerization of L-dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) in the melanin biosynthetic pathway. We have recently demonstrated that the constitutive expression of TYRP2 in human melanoma cells positively correlates with cis-diamminedichloroplatinum (II) (CDDP) resistance, and that the ectopic expression of TYRP2 in CDDP-sensitive cells rendered them more resistant to CDDP treatment. Here, we demonstrate that this correlation between constitutive TYRP2 expression and CDDP resistance applies to a panel of distinct human melanoma cell lines obtained from patients with melanoma at various stages of disease progression. We further show that CDDP resistance correlates only with TYRP2 expression and is associated neither with tyrosinase and TYRP1 expression, nor with cellular melanin content. Together, these results further support the notion that TYRP2 is a novel mediator of CDDP resistance in melanoma cells and suggest that this function of TYRP2 is independent of cellular melanin content and of the other regulatory enzymes of the melanogenic pathway.
Subject(s)
Cisplatin/toxicity , Drug Resistance, Neoplasm , Intramolecular Oxidoreductases/genetics , Melanins/metabolism , Membrane Glycoproteins , Oxidoreductases , Cell Survival/drug effects , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Intramolecular Oxidoreductases/metabolism , Melanoma , Monophenol Monooxygenase/genetics , Proteins/genetics , Tumor Cells, CulturedABSTRACT
Like other types of pre-malignant lesions and carcinoma, angiogenesis is associated with high-grade cervical dysplasia and with invasive squamous carcinoma of the cervix. Vascular endothelial cell growth factor (VEGF) is known to be one of the most important inducers of angiogenesis and is upregulated in carcinoma of the cervix. Human Papilloma Virus 16 (HPV-16) has been etiologically linked to human cervical cancer, and the major oncogenic proteins encoded by the viral genome, E6 and E7, are involved in the immortalization of target cells. Because several oncogenes including mutant ras, EGF receptor, ErbB2/Her2, c-myc and v-src upregulate VEGF expression, we asked whether HVP-16 E6 oncoprotein could act in a similar fashion. We found that HPV-16 E6-positive cells generally express high levels of VEGF message. Furthermore, co-expression of the VEGF promoter-Luc (luciferase) reporter gene with E6 in both human keratinocytes and mouse fibroblast showed that E6 oncoprotein upregulates VEGF promoter activity, and does so in a p53 independent manner. An E6 responsive region which comprises four Sp-1 sites, between -194 and -50 bp of the VEGF promoter, is also necessary for constitutive VEGF transcription. Taken together, our results suggest the possibility that the HPV oncoprotein E6 may contribute to tumor angiogenesis by direct stimulation of the VEGF gene.
Subject(s)
Endothelial Growth Factors/genetics , Genes, p53 , Lymphokines/genetics , Neovascularization, Pathologic/genetics , Oncogene Proteins, Viral/physiology , Papillomaviridae/genetics , Promoter Regions, Genetic , Repressor Proteins , Tumor Suppressor Protein p53/physiology , Autocrine Communication , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/virology , Endothelial Growth Factors/metabolism , ErbB Receptors/physiology , Female , HeLa Cells/metabolism , HeLa Cells/virology , Humans , Keratinocytes/virology , Lymphokines/metabolism , Neoplasm Proteins/physiology , Neovascularization, Pathologic/physiopathology , Oncogene Proteins, Viral/genetics , Papillomaviridae/physiology , Papillomavirus Infections/pathology , Papillomavirus Infections/virology , Recombinant Fusion Proteins/biosynthesis , Transcription, Genetic , Transcriptional Activation , Transforming Growth Factor alpha/physiology , Tumor Cells, Cultured/metabolism , Tumor Cells, Cultured/virology , Tumor Virus Infections/pathology , Tumor Virus Infections/virology , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/virology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Vulvar Neoplasms/metabolism , Vulvar Neoplasms/pathology , Vulvar Neoplasms/virologyABSTRACT
Almost all known conventional cytotoxic anticancer drugs are less effective in killing tumor cells grown as multicellular spheroids than in killing tumor cells grown as monolayer cell cultures. This "multicellular resistance" reflects the relative intrinsic drug-resistant phenotype of most solid tumors growing in vivo and is due to factors such as limited drug penetration or reduced fractions of proliferating cells. Proteasome inhibitors such as PS-341, a dipeptide boronic acid analogue, represent an interesting new class of potential anticancer drugs, which are entering early-phase clinical trials. PS-341 has been found to have good broad-spectrum cytotoxic activity in the 60-monolayer cell line National Cancer Institute screen. However, because its relative potency has not been tested in spheroid systems, we analyzed the activity of PS-341 in a spheroid/solid tumor context using four different human ovarian carcinoma cell lines and three prostate carcinoma cell lines, respectively. We found, with one exception, that PS-341 showed equal or greater activity in spheroids than in the respective monolayer cell cultures, even in a prostate cancer spheroid model with a very low growth fraction. PS-341 induced apoptotic cell death in carcinoma cells in both culture systems. We also noted a decrease in XIAP protein, a member of the inhibitor of apoptosis (IAP) family of apoptosis inhibitors, and phosphorylation of Bcl-XL in PS-341-treated ovarian carcinoma cells. Furthermore, DNA fragmentation, a hallmark of apoptosis (in this case, induced by PS-341), was completely inhibited by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD). Taken together, the results indicate that unlike most other known anticancer cytotoxic drugs, PS-341 appears to be as effective in killing tumor cells grown in the form of multicell spheroids as in killing tumor cells grown in monolayer cell culture. Hence, this compound has the potential to circumvent multicellular drug resistance and, as such, may show promising activity against solid tumors with low growth fractions in vivo, which are frequently intrinsically resistant to conventional cytotoxic anticancer drugs.
Subject(s)
Antineoplastic Agents/pharmacology , Boronic Acids/pharmacology , Ovarian Neoplasms/pathology , Prostatic Neoplasms/pathology , Protease Inhibitors/pharmacology , Pyrazines/pharmacology , Spheroids, Cellular/drug effects , Apoptosis/drug effects , Bortezomib , Carcinoma/drug therapy , Carcinoma/metabolism , Carcinoma/pathology , Caspase Inhibitors , Caspases/metabolism , Cell Adhesion/drug effects , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , Drug Resistance, Neoplasm , Female , Humans , Male , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Phosphorylation/drug effects , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Spheroids, Cellular/pathology , Tumor Cells, Cultured/drug effects , X-Linked Inhibitor of Apoptosis Protein , bcl-X ProteinABSTRACT
A number of drugs currently being tested in clinical trials as possible angiogenesis inhibitors were not originally developed with the intention of suppressing tumour angiogenesis. Thalidomide and interferon alpha are obvious examples of such drugs. This list of 'accidental' angiogenesis inhibitors may include established agents such as conventional cytotoxic chemotherapeutic drugs as well as the new generation of anticancer drugs known as anti-oncoprotein signal transduction inhibitors. With respect to the former, the potential of such drugs to inhibit angiogenesis could be the result of their ability to cause collateral damaging effects on cycling endothelial cells found in newly formed blood vessels, or inhibiting other vital endothelial cell functions necessary for angiogenesis. The antitumour vascular side-effects of chemotherapy may be optimised by administering such drugs continuously on a more frequent (e.g. weekly or even daily) basis at levels well below the maximum tolerated dose (MTD), especially when this is done in combination with newly developed anti-angiogenic drugs such as vascular endothelial cell growth factor (VEGF) receptor blocking antibodies. This strategy may minimise or delay the problems of host toxicity and acquired drug resistance. The possibility of anti-angiogenic effects mediated by signal transduction inhibitors such as ras farnesyltransferase inhibitors (ras FTI's), or drugs which block receptor tyrosine kinases (e.g. ErbB2/neu) such as Herceptin, may be the consequence of such oncogenes inducing or upregulating various pro-angiogenic molecules such as VEGF (vascular endothelial cell growth factor) in tumour cells. Hence, treatment of tumour cells with such drugs can lead to downregulation of tumour cell-associated VEGF expression and this can contribute to an anti-angiogenic effect of the drug in vivo. In addition, some of these drugs may also affect certain 'activated' endothelial cell functions directly so as to block angiogenesis. An awareness of the potential of such conventional or experimental anticancer drugs to affect tumour growth through blockade or suppression of angiogenesis has implications for how anticancer drugs may be used clinically, either alone, or in combination with other drugs to optimally treat cancer.