ABSTRACT
BACKGROUND: Novel anti-angiogenic treatments are increasingly complementing established cancer therapy strategies in head and neck tumors. Contrast-enhanced magnetic resonance imaging (MRI) can be applied for early and non-invasive therapy monitoring by non-invasive quantitative assessment of tumor microcirculation as in vivo imaging biomarkers of therapy response. PURPOSE: To monitor the anti-angiogenic effects of a novel combination therapy on experimental head and neck squamous cell carcinomas (HNSCC) with dynamic contrast-enhanced (DCE)-MRI. MATERIAL AND METHODS: Athymic rats (n = 18) with subcutaneous HNSCC xenografts were investigated by DCE-MRI before and after 7 days of a daily triple therapy regimen combining the COX-II-inhibitor celecoxib, the matrix-metalloproteinase-inhibitor GM6001, and the uPA-inhibitor upamostat. Quantitative measurements of tumor blood flow (tBF), tumor blood volume (tBV), and permeability-surface area product (PS) were calculated and validated by immunohistochemistry. RESULTS: Mean tBF and tBV in triple-therapy animals decreased significantly from day 0 to day 7 (tBF, 41.0 ± 14.2 to 20.4 ± 5.7 mL/100 mL/min; P < 0.01; tBV, 17.7 ± 3.9 to 7.5 ± 3.3%; P < 0.01). No significant effects on PS were observed in either group (P > 0.05). Immunohistochemical analysis showed a significantly lower tumor vascularity in the therapy group than in the control group (CD31), significantly fewer Ki-67+ proliferating tumor cells and significantly more Capase-3+ apoptotic tumor cells (P < 0.05). Significant (P < 0.05) correlations were observed between tBF/tBV and CD31 (tBF, r = 0.84; tBV, r = 0.70), tBV and Ki-67 (r = 0.62), as well as tBF and caspase-3 (r = -0.64). CONCLUSION: DCE-MRI may be a suitable tool for the non-invasive monitoring of the anti-vascular effects of this innovative triple therapy regimen with potential for clinical translation.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Biomarkers, Tumor/analysis , Carcinoma, Squamous Cell/chemistry , Carcinoma, Squamous Cell/drug therapy , Hypopharyngeal Neoplasms/chemistry , Hypopharyngeal Neoplasms/drug therapy , Image Enhancement/methods , Animals , Celecoxib , Combined Modality Therapy , Contrast Media , Dipeptides/therapeutic use , Disease Models, Animal , Immunohistochemistry/methods , Magnetic Resonance Imaging/methods , Oximes , Piperazines/therapeutic use , Pyrazoles/therapeutic use , Rats , Rats, Nude , Reproducibility of Results , Sulfonamides/therapeutic use , Xenograft Model Antitumor Assays/methodsABSTRACT
OBJECTIVES: To evaluate the use of diffusion-weighted MRI (DW-MRI) and volume measurements for early monitoring of antiangiogenic therapy in an experimental tumor model. MATERIALS AND METHODS: 23 athymic nude rats, bearing human colon carcinoma xenografts (HT-29) were examined before and after 6 days of treatment with regorafenib (nâ=â12) or placebo (nâ=â11) in a clinical 3-Tesla MRI. For DW-MRI, a single-shot EPI sequence with 9 b-values (10-800 s/mm2) was used. The apparent diffusion coefficient (ADC) was calculated voxelwise and its median value over a region of interest, covering the entire tumor, was defined as the tumor ADC. Tumor volume was determined using T2-weighted images. ADC and volume changes between first and second measurement were evaluated as classifiers by a receiver-operator-characteristic (ROC) analysis individually and combined using Fisher's linear discriminant analysis (FLDA). RESULTS: All ADCs and volumes are stated as median±standard deviation. Tumor ADC increased significantly in the therapy group (0.76±0.09×10(-3) mm2/s to 0.90±0.12×10(-3) mm2/s; p<0.001), with significantly higher changes of tumor ADC than in the control group (0.10±0.11×10(-3) mm2/s vs. 0.03±0.09×10(-3) mm2/s; pâ=â0.027). Tumor volume increased significantly in both groups (therapy: 347.8±449.1 to 405.3±823.6 mm3; pâ=â0.034; control: 219.7±79.5 to 443.7±141.5 mm3; p<0.001), however, the therapy group showed significantly reduced tumor growth (33.30±47.30% vs. 96.43±31.66%; p<0.001). Area under the curve and accuracy of the ADC-based ROC analysis were 0.773 and 78.3%; and for the volume change 0.886 and 82.6%. The FLDA approach yielded an AUC of 0.985 and an accuracy of 95.7%. CONCLUSIONS: Regorafenib therapy significantly increased tumor ADC after 6 days of treatment and also significantly reduced tumor growth. However, ROC analyses using each parameter individually revealed a lack of accuracy in discriminating between therapy and control group. The combination of both parameters using FLDA substantially improved diagnostic accuracy, thus highlighting the potential of multi-parameter MRI as an imaging biomarker for non-invasive early tumor therapy monitoring.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Carcinoma/pathology , Colonic Neoplasms/pathology , Diffusion Magnetic Resonance Imaging/methods , Heterografts/pathology , Phenylurea Compounds/therapeutic use , Pyridines/therapeutic use , Animals , Carcinoma/drug therapy , Colonic Neoplasms/drug therapy , Female , HT29 Cells , Heterografts/drug effects , Humans , Rats, Nude , Tumor BurdenABSTRACT
Dye-effluxing side population (SP) cells can be resistant to chemotherapy and are thought to resemble cancer stem cells. We characterized the relevance of the SP subpopulation in esophageal cancer cell lines and their relation to chemotherapy resistance and metastasis. The SP subpopulation was detected using Hoechst 33342 staining in five esophageal cancer cell lines OE19, OE21, OE33, PT1590, and LN1590. CTx-resistant cell lines were developed after long-term exposure to 5-fluorouracil (5-FU) and cisplatin and validated by analysis of resistance markers, thymidylate synthase and ERCC1. While neither LN1590 nor PT1590 had detectable SP cells, OE19, OE21, and OE33 cells were found to contain varying levels of SP cells. With increasing duration of 5-FU or cisplatin therapy, the SP subpopulation substantially emerged in PT1590 and LN1590. OE19-SP cells displayed significant higher tumorigenicity than OE19- non-SP (NSP) cells after subcutaneous tumor cell injection in vivo. SP cells isolated from OE19 and OE19/5-FUres were subsequently analyzed by an epithelial-to-mesenchymal transition (EMT) polymerase chain reaction array. Interestingly, the SP fraction of OE19/5-FUres showed a dramatic upregulation of EMT-related genes compared to the SP fraction of OE19. Our results provide evidence that (1) the proportion of SP cells is different in esophageal cancer, (2) SP cells exhibit stem cell properties and are associated to chemotherapy resistance, and (3) long-term CTx selects for SP cells with an upregulated EMT gene profile, which might be the source of systemic disease relapse. Further investigations are necessary to ideally target these EMT-associated SP cells in esophageal cancer.
Subject(s)
Carcinoma, Squamous Cell/pathology , Drug Resistance, Neoplasm , Esophageal Neoplasms/pathology , Neoplasm Metastasis/pathology , Neoplastic Stem Cells/cytology , Side-Population Cells/cytology , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/biosynthesis , Adenocarcinoma/drug therapy , Adenocarcinoma/pathology , Animals , Antimetabolites/pharmacology , Antineoplastic Agents/pharmacology , Carcinoma, Squamous Cell/drug therapy , Cell Differentiation , Cell Line, Tumor , Cisplatin/pharmacology , DNA-Binding Proteins/biosynthesis , Endonucleases/biosynthesis , Epithelial-Mesenchymal Transition , Esophageal Neoplasms/drug therapy , Esophageal Squamous Cell Carcinoma , Fluorouracil/pharmacology , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/biosynthesis , Neoplasm Proteins/biosynthesis , Thymidylate Synthase/biosynthesis , beta Catenin/biosynthesisABSTRACT
OBJECTIVE: To investigate dynamic contrast-enhanced computed tomography for monitoring the effects of regorafenib on experimental colon carcinomas in rats by quantitative assessments of tumor microcirculation parameters with immunohistochemical validation. MATERIALS AND METHODS: Colon carcinoma xenografts (HT-29) implanted subcutaneously in female athymic rats (nâ=â15) were imaged at baseline and after a one-week treatment with regorafenib by dynamic contrast-enhanced computed tomography (128-slice dual-source computed tomography). The therapy group (nâ=â7) received regorafenib daily (10 mg/kg bodyweight). Quantitative parameters of tumor microcirculation (plasma flow, mL/100 mL/min), endothelial permeability (PS, mL/100 mL/min), and tumor vascularity (plasma volume, %) were calculated using a 2-compartment uptake model. Dynamic contrast-enhanced computed tomography parameters were validated with immunohistochemical assessments of tumor microvascular density (CD-31), tumor cell apoptosis (TUNEL), and proliferation (Ki-67). RESULTS: Regorafenib suppressed tumor vascularity (15.7±5.3 to 5.5±3.5%; p<0.05) and tumor perfusion (12.8±2.3 to 8.8±2.9 mL/100 mL/min; pâ=â0.063). Significantly lower microvascular density was observed in the therapy group (CD-31; 48±10 vs. 113±25, p<0.05). In regorafenib-treated tumors, significantly more apoptotic cells (TUNEL; 11844±2927 vs. 5097±3463, p<0.05) were observed. Dynamic contrast-enhanced computed tomography tumor perfusion and tumor vascularity correlated significantly (p<0.05) with microvascular density (CD-31; râ=â0.84 and 0.66) and inversely with apoptosis (TUNEL; râ=â-0.66 and -0.71). CONCLUSIONS: Regorafenib significantly suppressed tumor vascularity (plasma volume) quantified by dynamic contrast-enhanced computed tomography in experimental colon carcinomas in rats with good-to-moderate correlations to an immunohistochemical gold standard. Tumor response biomarkers assessed by dynamic contrast-enhanced computed tomography may be a promising future approach to a more personalized and targeted cancer therapy.
Subject(s)
Carcinoma/drug therapy , Colonic Neoplasms/drug therapy , Drug Monitoring/methods , Heterografts/drug effects , Phenylurea Compounds/pharmacology , Pyridines/pharmacology , Animals , Capillary Permeability/drug effects , Female , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Microcirculation/drug effects , Rats , Tomography, X-Ray Computed/methodsABSTRACT
Simian virus 40 (SV40) is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1δ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1δ mutants exhibited a reduced kinase activity compared to wtCK1δ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1δ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1δ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1δ. To characterize the effects of CK1δ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1δ under the control of the whey acidic protein (WAP) gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T) mice. Both WAP-T mice as well as WAP-mutCK1δ/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1δ/WAP-T bi-transgenic animals. The reduced CK1δ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1δ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1δ/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA repair. Our data show that inactivating mutations in CK1δ impair SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo.
Subject(s)
Casein Kinase Idelta/genetics , Casein Kinase Idelta/metabolism , Cell Transformation, Viral/genetics , Mammary Neoplasms, Experimental/enzymology , Mammary Neoplasms, Experimental/pathology , Mutation , Simian virus 40/physiology , Animals , Antigens, Viral, Tumor/immunology , Casein Kinase Idelta/chemistry , Cell Line , Cell Line, Tumor , Disease Progression , Female , Gene Expression Regulation , Male , Mammary Glands, Animal/metabolism , Mammary Glands, Animal/pathology , Mammary Glands, Animal/virology , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/virology , Mice , Mice, Inbred BALB C , Mice, Transgenic , Milk Proteins/genetics , Models, Molecular , Phenotype , Phosphorylation , Promoter Regions, Genetic/genetics , Protein Structure, Tertiary , Simian virus 40/immunology , Survival AnalysisABSTRACT
The protein kinase family, one of the largest gene families in eukaryotes, plays an important role in regulating various cellular processes such as cell proliferation, cell death, cell cycle progression, differentiation and cell survival. Therefore, it is not surprising that the deregulation of many kinases is usually directly linked to cancer development. In all solid tumors, changes in protein kinase expression levels and activities, as well as alterations in the degree of posttranslational modifications can contribute to cancer development. Consequently, the identification of molecular targets and signaling pathways specific to cancer cells is becoming more and more important for cancer drug development and cancer therapies. Inhibition of various protein kinases has already been investigated in many pre-clinical and clinical trials targeting all stages of signal transduction, demonstrating promising results in cancer therapy. Conventional chemotherapeutics are often ineffective as well as harmful; hence a combination of both chemotherapeutics and protein kinase inhibitors may result in new and more successful therapeutic approaches. In this review we focus on protein kinases involved in different signaling pathways and their alterations in solid tumors.
Subject(s)
Antineoplastic Agents/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Antibodies, Monoclonal/therapeutic use , Humans , Neoplasms/drug therapy , Neoplasms/enzymology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Signal Transduction/drug effectsABSTRACT
Casein kinase 1 epsilon (CK1epsilon) is involved in various cellular processes, including cell growth, differentiation, and apoptosis, vesicle transport, and control of the circadian rhythm. Deregulation of CK1epsilon has been linked to neurodegenerative diseases and cancer. To better understand the cell type-specific functions of CK1epsilon, we determined its localization by immunhistochemistry in tissues of healthy, young adult BALB/c mice and in mammary tumors of SV40 T-antigen-transgenic mice. CK1epsilon expression was found to be highly regulated in normal tissues of endodermal, mesodermal, and ectodermal origin and in neoplastic tissue of mammary cancer. The data presented here give an overview of CK1epsilon reactivity in different organs under normal conditions and outline changes in its expression in mammary carcinomas. Our data suggest a cell/organ type-specific function of CK1epsilon and indicate that tumorigenic conversion of mammary glands in SV40 T-antigen-transgenic mice leads to downregulation of CK1epsilon. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
Subject(s)
Antigens, Viral, Tumor/genetics , Casein Kinase 1 epsilon/genetics , Mammary Neoplasms, Animal/virology , Mammary Neoplasms, Experimental/enzymology , Animals , Antigens, Polyomavirus Transforming/genetics , Cell Transformation, Neoplastic/genetics , Ectoderm/enzymology , Endoderm/enzymology , Female , Male , Mammary Glands, Animal/enzymology , Mammary Glands, Animal/physiology , Mammary Neoplasms, Animal/enzymology , Mammary Neoplasms, Experimental/genetics , Mesoderm/enzymology , Mice , Mice, Inbred BALB C , Mice, Transgenic , Organ SpecificityABSTRACT
In this study, we report on the discovery of isoxazole 1 as a potent dual inhibitor of p38alpha (IC(50) = 0.45 microM) and CK1delta (IC(50) = 0.23 microM). Because only a few effective small molecule inhibitors of CK1 have been described so far, we aimed to develop this structural class toward specific agents. Molecular modeling studies comparing p38alpha/CK1delta suggested an optimization strategy leading to design, synthesis, biological characterization, and SAR of highly potent compounds including 9 (IC(50) p38alpha = 0.006 microM; IC(50) CK1delta = 1.6 microM), 13 (IC(50) p38alpha = 2.52 microM; IC(50) CK1delta = 0.033 microM), 17 (IC(50) p38alpha = 0.019 microM; IC(50) CK1delta = 0.004 microM; IC(50) CK1epsilon = 0.073 microM), and 18 (CKP138) (IC(50) p38alpha = 0.041 microM; IC(50) CK1delta = 0.005 microM; IC(50) CK1epsilon = 0.447 microM) possessing differentiated specificity. Selected compounds were profiled over 76 kinases and evaluation of their cellular efficacy showed 18 (CKP138) to be a highly potent and dual-specific inhibitor of CK1delta and p38alpha.
Subject(s)
Casein Kinase Idelta/antagonists & inhibitors , Imidazoles/chemistry , Imidazoles/pharmacology , Isoxazoles/chemistry , Isoxazoles/pharmacology , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Binding, Competitive , Casein Kinase Idelta/chemistry , Casein Kinase Idelta/genetics , Casein Kinase Idelta/metabolism , Cell Line , Drug Discovery , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , Imidazoles/metabolism , Inhibitory Concentration 50 , Isoxazoles/metabolism , Mitogen-Activated Protein Kinase 14/chemistry , Mitogen-Activated Protein Kinase 14/metabolism , Models, Molecular , Molecular Sequence Data , Mutation , Protein Conformation , Rats , Trophoblasts/cytology , Trophoblasts/drug effectsABSTRACT
BACKGROUND: Casein kinase 1 delta (CK1delta) phosphorylates many key proteins playing important roles in such biological processes as cell growth, differentiation, apoptosis, circadian rhythm and vesicle transport. Furthermore, deregulation of CK1delta has been linked to neurodegenerative diseases and cancer. In this study, the cell specific distribution of CK1delta in various tissues and organs of young adult BALB/c mice was analysed by immunohistochemistry. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical staining of CK1delta was performed using three different antibodies against CK1delta. A high expression of CK1delta was found in a variety of tissues and organ systems and in several cell types of endodermal, mesodermal and ectodermal origin. CONCLUSIONS: These results give an overview of the cell-type specific expression of CK1delta in different organs under normal conditions. Thus, they provide evidence for possible cell-type specific functions of CK1delta, where CK1delta can interact with and modulate the activity of key regulator proteins by site directed phosphorylation. Furthermore, they provide the basis for future analyses of CK1delta in these tissues.
Subject(s)
Casein Kinase Idelta/metabolism , Mice, Inbred BALB C/metabolism , Animals , Antibodies, Monoclonal , Immunohistochemistry , Mice , Mice, Inbred BALB C/anatomy & histology , Rabbits , Tissue DistributionABSTRACT
The involvement of CK1 (casein kinase 1) delta in the regulation of multiple cellular processes implies a tight regulation of its activity on many different levels. At the protein level, reversible phosphorylation plays an important role in modulating the activity of CK1delta. In the present study, we show that PKA (cAMP-dependent protein kinase), Akt (protein kinase B), CLK2 (CDC-like kinase 2) and PKC (protein kinase C) alpha all phosphorylate CK1delta. PKA was identified as the major cellular CK1deltaCK (CK1delta C-terminal-targeted protein kinase) for the phosphorylation of CK1delta in vitro and in vivo. This was implied by the following evidence: PKA was detectable in the CK1deltaCK peak fraction of fractionated MiaPaCa-2 cell extracts, PKA shared nearly identical kinetic properties with those of CK1deltaCK, and both PKA and CK1deltaCK phosphorylated CK1delta at Ser370 in vitro. Furthermore, phosphorylation of CK1delta by PKA decreased substrate phosphorylation of CK1delta in vitro. Mutation of Ser370 to alanine increased the phosphorylation affinity of CK1delta for beta-casein and the GST (gluthatione S-transferase)-p53 1-64 fusion protein in vitro and enhanced the formation of an ectopic dorsal axis during Xenopus laevis development. Anchoring of PKA and CK1delta to centrosomes was mediated by AKAP (A-kinase-anchoring protein) 450. Interestingly, pre-incubation of MiaPaCa-2 cells with the synthetic peptide St-Ht31, which prevents binding between AKAP450 and the regulatory subunit RII of PKA, resulted in a 6-fold increase in the activity of CK1delta. In summary, we conclude that PKA phosphorylates CK1delta, predominantly at Ser370 in vitro and in vivo, and that site-specific phosphorylation of CK1delta by PKA plays an important role in modulating CK1delta-dependent processes.