ABSTRACT
Pain is the most unbearable symptom accompanying primary bone cancers and bone metastases. Bone resorptive disorders are often associated with hypercalcemia, contributing to the pathologic process. Nitrogen-containing bisphosphonates (NBPs) are efficiently used to treat bone cancers and metastases. Apart from their toxic effect on cancer cells, NBPs also provide analgesia via poorly understood mechanisms. We previously showed that NBPs, by inhibiting the mevalonate pathway, induced formation of novel ATP analogs such as ApppI [1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) triphosphoric acid diester], which can potentially be involved in NBP analgesia. In this study, we used the patch-clamp technique to explore the action of ApppI on native ATP-gated P2X receptors in rat sensory neurons and rat and human P2X3, P2X2, and P2X7 receptors expressed in human embryonic kidney cells. We found that although ApppI has weak agonist activity, it is a potent inhibitor of P2X3 receptors operating in the nanomolar range. The inhibitory action of ApppI was completely blocked in hypercalcemia-like conditions and was stronger in human than in rat P2X3 receptors. In contrast, P2X2 and P2X7 receptors were insensitive to ApppI, suggesting a high selectivity of ApppI for the P2X3 receptor subtype. NBP, metabolite isopentenyl pyrophosphate, and endogenous AMP did not exert any inhibitory action, indicating that only intact ApppI has inhibitory activity. Ca2+-dependent inhibition was stronger in trigeminal neurons preferentially expressing desensitizing P2X3 subunits than in nodose ganglia neurons, which also express nondesensitizing P2X2 subunits. Altogether, we characterized previously unknown purinergic mechanisms of NBP-induced metabolites and suggest ApppI as the endogenous pain inhibitor contributing to cancer treatment with NBPs.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Calcium/pharmacology , Ion Channel Gating/drug effects , Purinergic P2X Receptor Antagonists/pharmacology , Receptors, Purinergic P2X3 , Adenosine Triphosphate/pharmacology , Animals , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Ion Channel Gating/physiology , Male , Rats , Rats, Wistar , Receptors, Purinergic P2X3/physiologyABSTRACT
INTRODUCTION: The immune system plays a major role in cancer progression. In solid tumors, 5-40 % of the tumor mass consists of tumor-associated macrophages (TAMs) and there is usually a correlation between the number of TAMs and poor prognosis, depending on the tumor type. TAMs usually resemble M2 macrophages. Unlike M1-macrophages which have pro-inflammatory and anti-cancer functions, M2-macrophages are immunosuppressive, contribute to the matrix-remodeling, and hence favor tumor growth. The role of TAMs is not fully understood in breast cancer progression. METHODS: Macrophage infiltration (CD68) and activation status (HLA-DRIIα, CD163) were evaluated in a large cohort of human primary breast tumors (562 tissue microarray samples), by immunohistochemistry and scored by automated image analysis algorithms. Survival between groups was compared using the Kaplan-Meier life-table method and a Cox multivariate proportional hazards model. Macrophage education by breast cancer cells was assessed by ex vivo differentiation of peripheral blood mononuclear cells (PBMCs) in the presence or absence of breast cancer cell conditioned media (MDA-MB231, MCF-7 or T47D cell lines) and M1 or M2 inducing cytokines (respectively IFN-γ, IL-4 and IL-10). Obtained macrophages were analyzed by flow cytometry (CD14, CD16, CD64, CD86, CD200R and CD163), ELISA (IL-6, IL-8, IL-10, monocyte colony stimulating factor M-CSF) and zymography (matrix metalloproteinase 9, MMP-9). RESULTS: Clinically, we found that high numbers of CD163(+) M2-macrophages were strongly associated with fast proliferation, poor differentiation, estrogen receptor negativity and histological ductal type (p<0.001) in the studied cohort of human primary breast tumors. We demonstrated ex vivo that breast cancer cell-secreted factors modulate macrophage differentiation toward the M2 phenotype. Furthermore, the more aggressive mesenchymal-like cell line MDA-MB231, which secretes high levels of M-CSF, skews macrophages toward the more immunosuppressive M2c subtype. CONCLUSIONS: This study demonstrates that human breast cancer cells influence macrophage differentiation and that TAM differentiation status correlates with recurrence free survival, thus further emphasizing that TAMs can similarly affect therapy efficacy and patient outcome.
Subject(s)
Breast Neoplasms/pathology , Leukocytes, Mononuclear/pathology , Macrophages/pathology , Breast Neoplasms/metabolism , Cell Differentiation/physiology , Cell Line, Tumor , Cell Proliferation/physiology , Cytokines/metabolism , Female , Humans , Leukocytes, Mononuclear/metabolism , Macrophage Colony-Stimulating Factor/metabolism , Macrophages/metabolism , Matrix Metalloproteinase 9/metabolismABSTRACT
BACKGROUND: Tumour stromal macrophages differentiate to tumour-associated macrophages (TAMs) with characteristics of immunosuppressive M2-type macrophages, having a central role in promoting tumour vascularisation, cancer cell dissemination and in suppressing anti-cancer immune responses. Bisphosphonates (BPs) are a group of drugs commonly used as anti-resorptive agents. Further, nitrogen containing BPs like Zoledronate (ZOL), are known to cause unspecific inflammatory reactions hence the hypothesis that its use could modulate TAMs polarization toward a more inflammatory phenotype. METHODS: We studied the in vitro polarization of J774 murine macrophages upon culture in 4T1 breast cancer cell-conditioned medium (4T1CM) and stimulation with LPS and free and liposome-encapsulated bisphosphonates. RESULTS: In this system, breast cancer soluble factors reduced the pro-inflammatory activation of macrophages but increased the secretion of matrix metalloproteinases (MMPs). In the presence of 4T1CM, a non-cytotoxic dose of liposome-encapsulated ZOL (ZOL-LIP) enhanced the expression of iNOS and TNF-α, markers of M1 activation, but did not diminish the expression of M2-type markers. In contrast, clodronate treatment either as a free drug (CLO) or liposome-encapsulated (CLO-LIP) decreased the expression of the M1-type markers and was highly cytotoxic to the macrophages. CONCLUSIONS: Breast cancer cells soluble factors modulate macrophages toward M2 activation state. Bisphosphonates may be applied to counteract this modulation. We propose that ZOL-LIP may be suitable for favouring cytotoxic immune responses by TAMs in breast cancer, whereas CLO-LIP may be appropriate for TAM depletion.
Subject(s)
Breast Neoplasms/metabolism , Culture Media, Conditioned/pharmacology , Diphosphonates/pharmacology , Imidazoles/pharmacology , Macrophages/drug effects , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Female , Humans , Liposomes , Macrophages/cytology , Macrophages/immunology , Matrix Metalloproteinases/metabolism , Mice , Nitric Oxide Synthase Type II/metabolism , Tumor Necrosis Factor-alpha/metabolism , Zoledronic AcidABSTRACT
Most human γδ T cells express Vγ2Vδ2 TCRs and play important roles in microbial and tumor immunity. Vγ2Vδ2 T cells are stimulated by self- and foreign prenyl pyrophosphate intermediates in isoprenoid synthesis. However, little is known about the molecular basis for this stimulation. We find that a mAb specific for butyrophilin 3 (BTN3)/CD277 Ig superfamily proteins mimics prenyl pyrophosphates. The 20.1 mAb stimulated Vγ2Vδ2 T cell clones regardless of their functional phenotype or developmental origin and selectively expanded blood Vγ2Vδ2 T cells. The γδ TCR mediates 20.1 mAb stimulation because IL-2 is released by ß(-) Jurkat cells transfected with Vγ2Vδ2 TCRs. 20.1 stimulation was not due to isopentenyl pyrophosphate (IPP) accumulation because 20.1 treatment of APC did not increase IPP levels. In addition, stimulation was not inhibited by statin treatment, which blocks IPP production. Importantly, small interfering RNA knockdown of BTN3A1 abolished stimulation by IPP that could be restored by re-expression of BTN3A1 but not by BTN3A2 or BTN3A3. Rhesus monkey and baboon APC presented HMBPP and 20.1 to human Vγ2Vδ2 T cells despite amino acid differences in BTN3A1 that localize to its outer surface. This suggests that the conserved inner and/or top surfaces of BTN3A1 interact with its counterreceptor. Although no binding site exists on the BTN3A1 extracellular domains, a model of the intracellular B30.2 domain predicts a basic pocket on its binding surface. However, BTN3A1 did not preferentially bind a photoaffinity prenyl pyrophosphate. Thus, BTN3A1 is required for stimulation by prenyl pyrophosphates but does not bind the intermediates with high affinity.
Subject(s)
Antigens, CD/immunology , Antigens, CD/metabolism , Hemiterpenes/immunology , Organophosphorus Compounds/immunology , T-Lymphocytes/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antigens, CD/genetics , Binding Sites, Antibody , Butyrophilins , Cell Line , HeLa Cells , Humans , Interleukin-2/metabolism , Lymphocyte Activation , Macaca mulatta , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Membrane Glycoproteins/metabolism , Membrane Proteins/immunology , Molecular Sequence Data , Papio , RNA Interference , RNA, Small Interfering , Receptors, Antigen, T-Cell, gamma-delta/immunology , Sequence Alignment , T-Lymphocytes/immunology , TerpenesABSTRACT
Human peripheral Vγ9Vδ2 T cells are activated by phosphorylated metabolites (phosphoagonists [PAg]) of the mammalian mevalonate or the microbial desoxyxylulose-phosphate pathways accumulated by infected or metabolically distressed cells. The underlying mechanisms are unknown. We show that treatment of nonsusceptible target cells with antibody 20.1 against CD277, a member of the extended B7 superfamily related to butyrophilin, mimics PAg-induced Vγ9Vδ2 T-cell activation and that the Vγ9Vδ2 T-cell receptor is implicated in this effect. Vγ9Vδ2 T-cell activation can be abrogated by exposing susceptible cells (tumor and mycobacteria-infected cells, or aminobisphosphonate-treated cells with up-regulated PAg levels) to antibody 103.2 against CD277. CD277 knockdown and domain-shuffling approaches confirm the key implication of the CD277 isoform BTN3A1 in PAg sensing by Vγ9Vδ2 T cells. Fluorescence recovery after photobleaching (FRAP) experiments support a causal link between intracellular PAg accumulation, decreased BTN3A1 membrane mobility, and ensuing Vγ9Vδ2 T-cell activation. This study demonstrates a novel role played by B7-like molecules in human γδ T-cell antigenic activation and paves the way for new strategies to improve the efficiency of immunotherapies using Vγ9Vδ2 T cells.
Subject(s)
Antigens, CD/metabolism , Antigens/metabolism , Lymphocyte Activation , Receptors, Antigen, T-Cell/metabolism , T-Lymphocyte Subsets/metabolism , Antibodies, Blocking , Antibodies, Immobilized , Antibodies, Monoclonal , Antigens, CD/chemistry , Antigens, CD/genetics , Butyrophilins , Cells, Cultured , Clone Cells , Enzyme Inhibitors/pharmacology , HEK293 Cells , Humans , Immunologic Factors/pharmacology , Lymphocyte Activation/drug effects , Phosphorylation/drug effects , Protein Isoforms/agonists , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Processing, Post-Translational/drug effects , Protein Transport/drug effects , RNA, Small Interfering , Receptors, Antigen, T-Cell/agonists , Receptors, Antigen, T-Cell/antagonists & inhibitors , Recombinant Proteins/agonists , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunologyABSTRACT
Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C-methyl-d-erythritol-4-phosphate pathway used by microbes, and isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Aminobisphosphonates and alkylamines indirectly stimulate Vγ2Vδ2 cells by inhibiting farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, thereby increasing IPP/triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester that directly stimulate. In this study, we further characterize stimulation by these compounds and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of Vγ2Vδ2 cells by aminobisphosphonates and alkylamines was much more sensitive to statin inhibition than stimulation by prenyl pyrophosphates; however, the continuous presence of aminobisphosphonates was toxic for T cells and blocked their proliferation. Aminobisphosphonate stimulation was rapid and prolonged, independent of known Ag-presenting molecules, and resistant to fixation. New classes of stimulatory compounds-mevalonate, the alcohol of HMBPP, and alkenyl phosphonates-likely stimulate differently. Mevalonate, a rate-limiting metabolite, appears to enter cells to increase IPP levels, whereas the alcohol of HMBPP and alkenyl phosphonates are directly recognized. The critical chemical feature of bisphosphonates is the amino moiety, because its loss switched aminobisphosphonates to direct Ags. Transfection of APCs with small interfering RNA downregulating FDPS rendered them stimulatory for Vγ2Vδ2 cells and increased cellular IPP. Small interfering RNAs for isopentenyl diphosphate isomerase functioned similarly. Our results show that a variety of manipulations affecting isoprenoid metabolism lead to stimulation of Vγ2Vδ2 T cells and that pulsing aminobisphosphonates would be more effective for the ex vivo expansion of Vγ2Vδ2 T cells for adoptive cancer immunotherapy.
Subject(s)
Lymphocyte Activation/immunology , Receptors, Antigen, T-Cell, gamma-delta/biosynthesis , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Terpenes/metabolism , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Cell Proliferation/drug effects , Clone Cells , Diphosphonates/metabolism , Diphosphonates/toxicity , Dose-Response Relationship, Immunologic , Growth Inhibitors/metabolism , Growth Inhibitors/pharmacology , Humans , Lymphocyte Activation/drug effects , Mevalonic Acid/metabolism , Mevalonic Acid/toxicity , Signal Transduction/drug effects , Signal Transduction/immunology , T-Lymphocyte Subsets/drug effects , Terpenes/toxicityABSTRACT
BACKGROUND: Tumour associated macrophages (TAMs) are important players in breast tumour progression and metastasis. Clinical and preclinical evidence suggests a role for zoledronate (ZOL) in breast cancer metastasis prevention. Further, zoledronate is able to induce inflammatory activation of monocytes and macrophages, which can be favourable in cancer treatments. The inherent bone tropism of zoledronate limits its availability in soft tissues and tumours. In this study we utilised an orthotopic murine breast cancer model to evaluate the possibility to use liposomes (EMP-LIP) to target zoledronate to tumours to modify TAM activation. METHODS: Triple-negative breast cancer 4T1 cells were inoculated in the 4th mammary fat pad of female Balb/c mice. Animals were divided according to the treatment: vehicle, ZOL, EMP-LIP and liposome encapsulated zoledronate (ZOL-LIP). Treatment was done intravenously (with tumour resection) and intraperitoneally (without tumour resection). Tumour growth was followed by bioluminescence in vivo imaging (IVIS) and calliper measurements. Tumour-infiltrating macrophages were assessed by immunohistochemical and immunofluorescence staining. Protein and RNA expression levels of inflammatory transcription factors and cytokines were measured by Western Blotting and Taqman RT-qPCR. RESULTS: Liposome encapsulated zoledronate (ZOL-LIP) treatment suppressed migration of 4T1 cell in vitro. Tumour growth and expression of the angiogenic marker CD34 were reduced upon both ZOL and ZOL-LIP treatment in vivo. Long-term ZOL-LIP treatment resulted in shift towards M1-type macrophage polarization, increased CD4 T cell infiltration and activation of NF-κB indicating changes in intratumoural inflammation, whereas ZOL treatment showed similar but non-significant trends. Moreover, ZOL-LIP had a lower bisphosphonate accumulation in bone compared to free ZOL. CONCLUSION: Results show that the decreased bisphosphonate accumulation in bone promotes the systemic anti-tumour effect of ZOL-LIP by increasing inflammatory response in TNBC tumours via M1-type macrophage activation.
Subject(s)
Liposomes , Triple Negative Breast Neoplasms , Humans , Female , Mice , Animals , Zoledronic Acid/pharmacology , Liposomes/pharmacology , Triple Negative Breast Neoplasms/drug therapy , Cell Line, Tumor , Diphosphonates/therapeutic use , Diphosphonates/pharmacology , Macrophages , Mice, Inbred BALB CABSTRACT
PURPOSE: For nanocarrier-based targeted delivery systems, preventing phagocytosis for prolong circulation half life is a crucial task. PEGylated poly(n-butylcyano acrylate) (PBCA) NP has proven a promising approach for drug delivery, but an easy and reliable method of PEGylation of PBCA has faced a major bottleneck. METHODS: PEGylated PBCA NPs containing docetaxel (DTX) by modified anionic polymerization reaction in aqueous acidic media containing amine functional PEG were made as an single step PEGylation method. In vitro colloidal stability studies using salt aggregation method and antiopsonization property of prepared NPs using mouse macrophage cell line RAW264 were performed. In vitro performance of anticancer activity of prepared formulations was checked on MCF7 cell line. NPs were radiolabeled with 99mTc and intravenously administered to study blood clearance and biodistribution in mice model. RESULTS: These formulations very effectively prevented phagocytosis and found excellent carrier for drug delivery purpose. In vivo studies display long circulation half life of PBCA-PEG20 NP in comparison to other formulations tested. CONCLUSIONS: The PEGylated PBCA formulation can work as a novel tool for drug delivery which can prevent RES uptake and prolong circulation half life.
Subject(s)
Drug Carriers/pharmacokinetics , Enbucrilate/pharmacokinetics , Nanoparticles , Polyethylene Glycols/pharmacokinetics , Animals , Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Cell Line , Cell Line, Tumor , Docetaxel , Drug Carriers/chemistry , Enbucrilate/chemistry , Female , Half-Life , Humans , Macrophages/metabolism , Mice , Phagocytosis/drug effects , Polyethylene Glycols/chemistry , Taxoids/administration & dosage , Technetium/chemistry , Tissue DistributionABSTRACT
Bisphosphonates (BPs) are potent inhibitors of osteoclast function, widely used to treat excessive bone resorption associated with bone metastases, that also have anti-tumor activity. Zoledronic acid (ZOL) represents a potential chemotherapeutic agent for the treatment of cancer. ZOL is the most potent nitrogen-containing BPs, and it inhibits cell growth and induces apoptosis in a variety of cancer cells. Recently we demonstrated that accumulation of isopentenyl pyrophosphate and the consequent formation of a new type of ATP analog (ApppI) after mevalonate pathway inhibition by nitrogen-containing BPs strongly correlates with ZOL-induced cell death in cancer cells in vitro. In this study we show that ZOL-induced apoptosis in HF28RA human follicular lymphoma cells occurs exclusively via the mitochondrial pathway, involves lysosomes, and is dependent on mevalonate pathway inhibition. To define the exact signaling pathway connecting them, we used modified HF28RA cell lines overexpressing either BclXL or dominant-negative caspase-9. In both mutant cells, mitochondrial and lysosomal membrane permeabilization (MMP and LMP) were totally prevented, indicating signaling between lysosomes and mitochondria and, additionally, an amplification loop for MMP and/or LMP regulated by caspase-9 in association with farnesyl pyrophosphate synthetase inhibition. Additionally, the lysosomal pathway in ZOL-induced apoptosis plays an additional/amplification role of the intrinsic pathway independently of caspase-3 activation. Moreover, we show a potential regulation by Bcl-XL and caspase-9 on cell cycle regulators of S-phase. Our findings provide a molecular basis for new strategies concomitantly targeting cell death pathways from multiple sites.
Subject(s)
Apoptosis/drug effects , Diphosphonates/pharmacology , Imidazoles/pharmacology , Lymphoma, Follicular/pathology , Lysosomes/metabolism , Mitochondria/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Animals , Caspase Inhibitors , Cathepsins/antagonists & inhibitors , Cattle , Cell Line, Tumor , Cell Membrane Permeability/drug effects , Hemiterpenes/metabolism , Humans , Lysosomes/drug effects , Mevalonic Acid/metabolism , Mice , Mitochondria/drug effects , Organophosphorus Compounds/metabolism , Prenylation/drug effects , Protease Inhibitors/pharmacology , Signal Transduction/drug effects , Zoledronic Acid , bcl-X Protein/metabolismABSTRACT
A class of drugs successfully used for treatment of metabolic bone diseases is the nitrogen-containing bisphosphonates (N-BPs), which act by inhibiting the vital enzyme, farnesyl pyrophosphate synthase (FPPS), of the mevalonate pathway. Inhibition of FPPS by N-BPs results in the intracellular accumulation of isopentenyl pyrophosphate (IPP) and consequently induces the biosynthesis of a cytotoxic ATP analog (ApppI). Previous cell-free data has reported that N-BPs inhibit FPPS by time-dependent manner as a result of the conformational change. This associated conformational change can be measured as an isomerization constant (K(isom)) and reflects the binding differences of the N-BPs to FPPS. In the present study, we tested the biological relevance of the calculated K(isom) values of zoledronic acid, risedronate and five experimental N-BP analogs in the cell culture model. We used IPP/ApppI formation as a surrogate marker for blocking of FPPS in the mevalonate pathway. As a result, a correlation between the time-dependent inhibition of FPPS and IPP/ApppI formation by N-BPs was observed. This outcome indicates that the time-dependent inhibition of FPPS enzyme is a biologically significant mechanism and further supports the use of the K(isom) calculations for evaluation of the overall potency of the novel FPPS inhibitors. Additionally, data illustrates that IPP/ApppI analysis is a useful method to monitor the intracellular action of drugs and drug candidates based on FPPS inhibition.
Subject(s)
Dimethylallyltranstransferase/antagonists & inhibitors , Diphosphonates/pharmacology , Mevalonic Acid/metabolism , Cells, Cultured , Dimethylallyltranstransferase/chemistry , Diphosphonates/chemistry , Humans , Nitrogen/chemistry , Time FactorsABSTRACT
Hepatocellular carcinoma (HCC) and colorectal carcinoma with hepatic metastases (mCRC) are cancers with poor prognosis and limited therapeutic options. New approaches are needed and adoptive immunotherapy with Vgamma9Vdelta2 T lymphocytes represents an attractive strategy. Indeed, Vgamma9Vdelta2 T cells were shown to exhibit efficient lytic activity against various human tumor cell lines, and in vitro Vgamma9Vdelta2 T expansion protocol based on single phosphoantigen stimulation could be easily performed for healthy donors. However, a low proliferative response of Vgamma9Vdelta2 T cells was observed in about half of the cancer patients, leading to an important limitation in the development of Vgamma9Vdelta2 T cell-based immunotherapy. Here, for the first time in the context of cancer patients, Vgamma9Vdelta2 T cell expansions were performed by co-culturing peripheral blood mononuclear cell (PBMCs) with autologous dendritic cells (DCs) pretreated with aminobisphosphonate zoledronate. For patients not responding to the conventional culture protocol, co-culture of PBMC with zoledronate-pretreated DCs induced strong cell expansion and allowed reaching a minimal rate of purity of 70% of Vgamma9Vdelta2 T cells. The potent immunostimulatory activity of zoledronate-treated DCs was associated with higher amount of isopentenyl pyrophosphate (IPP) in the culture and was correlated with better ability to activate Vgamma9Vdelta2 T cells as measured by IFN-gamma production. Moreover, we demonstrated that the cytotoxic level of Vgamma9Vdelta2 T cells against freshly autologous tumor cells isolated from patients could be significantly increased by pretreating the tumor cells with zoledronate. Thus, this method of generating Vgamma9Vdelta2 T cells leads eligible for Vgamma9Vdelta2 T cell adoptive immunotherapy the HCC and mCRC patients.
Subject(s)
Colorectal Neoplasms/therapy , Dendritic Cells/immunology , Diphosphonates/pharmacology , Imidazoles/pharmacology , Immunotherapy, Adoptive , Liver Neoplasms/therapy , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Adenocarcinoma/immunology , Adenocarcinoma/pathology , Adenocarcinoma/therapy , Aged , Blotting, Western , Bone Density Conservation Agents/pharmacology , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/therapy , Cell Differentiation , Cell Proliferation , Coculture Techniques , Colorectal Neoplasms/immunology , Colorectal Neoplasms/pathology , Cytotoxicity, Immunologic , Dendritic Cells/drug effects , Diphosphates/pharmacology , Female , Flow Cytometry , Hemiterpenes/metabolism , Humans , Liver Neoplasms/immunology , Liver Neoplasms/secondary , Male , Middle Aged , Organophosphorus Compounds/metabolism , Receptors, Antigen, T-Cell, gamma-delta/immunology , T-Lymphocyte Subsets/immunology , Zoledronic AcidABSTRACT
P-Glycoprotein mediated efflux is one of the barriers limiting drug absorption from the intestine. Predictions of the intestinal P-glycoprotein function need to take into account the concentration dependency because high intestinal drug concentrations may saturate P-glycoprotein. However, the substrate binding site of P-glycoprotein lies inside the cells and the drug concentration at the binding site cannot be measured directly. Therefore, rigorous determination of concentration dependent P-glycoprotein kinetics is challenging. In this study, the effects of the aqueous boundary layers, extracellular pH and cellular retention on the apparent saturation kinetics of P-glycoprotein mediated transport of quinidine in an in vitro cell permeation setting were explored. The changes in the experimental conditions caused 1 order of magnitude variation in the apparent affinity to P-glycoprotein (K(m,app)) and a 5-fold difference in the maximum effective P-glycoprotein mediated transport rate of quinidine (V(max,app)). However, fitting the concentration data into a compartmental model which accounted for the aqueous boundary layers, cell membranes and cellular retention suggested that the P-glycoprotein function per se was not altered, it was the differences in the passive transfer of quinidine which changed the apparent transport kinetics. These results provide further insight into the dynamics of the P-glycoprotein mediated transport and on the roles of several confounding factors involved in in vitro experimental setting. Further, the results confirm the applicability of compartmental model based data analysis approach in the determination of active transporter kinetics.
Subject(s)
Biological Transport, Active/physiology , Quinidine/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Biological Transport , Caco-2 Cells , Cell Line , Dogs , Humans , KineticsABSTRACT
Beta amyloid plays a main role in the pathophysiology of Alzheimer's disease by inducing oxidative stress in the brain. Curcumin, a natural antioxidant, is known to inhibit beta amyloid and beta amyloid induced oxidative stress. However, low bioavailability and photodegradation are the major concerns for the use of curcumin. In the present study, we have formulated apolipoprotein E3 mediated poly(butyl) cyanoacrylate nanoparticles containing curcumin (ApoE3-C-PBCA) to provide photostability and enhanced cell uptake of curcumin by targeting. Prepared nanoparticles were characterized for particle size, zeta potential, entrapment efficiency and in vitro drug release. The entrapment of curcumin inside the nanoparticles was confirmed by X-ray diffraction analysis. Physicochemical characterization confirmed the suitability of the method of preparation. The photostability of curcumin was increased significantly in nanoparticles compared to plain curcumin. In vitro cell culture study showed enhanced therapeutic efficacy of ApoE3-C-PBCA against beta amyloid induced cytotoxicity in SH-SY5Y neuroblastoma cells compared to plain curcumin solution. Beta amyloid is known to induce apoptosis in neuronal cells, therefore antiapoptotic activity of curcumin was studied using flow cytometry assays. From all the experiments, it was found that the activity of curcumin was enhanced with ApoE3-C-PBCA compared to plain curcumin solution suggesting enhanced cell uptake and a sustained drug release effect. The synergistic effect of ApoE3 and curcumin was also studied, since ApoE3 also possesses both antioxidant and antiamyloidogenic activity. It was found that ApoE3 did indeed have activity against beta amyloid induced cytotoxicity along with curcumin. Hence, ApoE3-C-PBCA offers great advantage in the treatment of beta amyloid induced cytotoxicity in Alzheimer's disease.
Subject(s)
Amyloid beta-Peptides/metabolism , Apolipoprotein E3/chemistry , Curcumin/chemistry , Curcumin/therapeutic use , Enbucrilate/chemistry , Polymers/chemistry , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Amyloid beta-Peptides/physiology , Apoptosis/drug effects , Caspase 3/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Flow Cytometry , Humans , Models, Theoretical , Nanoparticles/chemistry , Reactive Oxygen Species/metabolism , X-Ray DiffractionABSTRACT
After decades of successful clinical use, the exact molecular mechanisms by which the anti-resorptive bisphosphonate drugs (BPs) exert their effects are now being revealed. In addition to their anti-resorptive effects, it is now apparent that nitrogen-containing BPs (N-BPs) have immunomodulatory properties. Specifically, these drugs activate immune cells called gamma, delta T lymphocytes. In this chapter we discuss the mechanism of gamma, delta T cell activation by N-BPs and propose that N-BPs may provide a safe and effective means for manipulating gamma,delta T cell activity in future immunotherapeutic approaches.
Subject(s)
Diphosphonates/pharmacology , Lymphocyte Activation/drug effects , Receptors, Antigen, T-Cell, gamma-delta/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Acute-Phase Reaction/immunology , Animals , Cell Proliferation/drug effects , Diphosphonates/chemistry , MiceABSTRACT
Bisphosphonates (BP) are a class of calcium-binding drug used to prevent bone resorption in skeletal disorders such as osteoporosis and metastatic bone disease. They act by selectively targeting bone-resorbing osteoclasts and can be grouped into two classes depending on their intracellular mechanisms of action. Simple BPs cause osteoclast apoptosis after cytoplasmic conversion into toxic ATP analogues. In contrast, nitrogen-containing BPs potently inhibit FPP synthase, an enzyme of the mevalonate (cholesterol biosynthesis) pathway. This results in production of a toxic metabolite (ApppI) and the loss of long-chain isoprenoid lipids required for protein prenylation, a process necessary for the function of small GTPase proteins essential for the survival and activity of osteoclasts. In this review we provide a state-of-the-art overview of these mechanisms of action and a historical perspective of how they were discovered. Finally, we challenge the long-held dogma that BPs act only in the skeleton and highlight recent studies that reveal insights into hitherto unknown effects on tumour-associated and tissue-resident macrophages.
Subject(s)
Bone Resorption , Diphosphonates , Bone Resorption/drug therapy , Bone and Bones , Diphosphonates/pharmacology , Humans , Osteoclasts , Protein PrenylationABSTRACT
Nitrogen-containing bisphosphonates indirectly activate Vgamma9Vdelta2 T cells through inhibition of farnesyl pyrophosphate synthase and intracellular accumulation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), but the cells responsible for Vgamma9Vdelta2 T cell activation through IPP/DMAPP accumulation are unknown. Treatment of human peripheral blood mononuclear cells (PBMCs) with a pharmacologically relevant concentration of zoledronic acid induced accumulation of IPP/DMAPP selectively in monocytes, which correlated with efficient drug uptake by these cells. Furthermore, zoledronic acid-pulsed monocytes triggered activation of gammadelta T cells in a cell contact-dependent manner. These observations identify monocytes as the cell type directly affected by bisphosphonates responsible for Vgamma9Vdelta2 T cell activation.
Subject(s)
Diphosphonates/pharmacology , Hemiterpenes/metabolism , Imidazoles/pharmacology , Monocytes/immunology , Organophosphorus Compounds/metabolism , Receptors, Antigen, T-Cell, gamma-delta/metabolism , T-Lymphocytes/metabolism , Cell Communication , Cells, Cultured , Humans , Lymphocyte Activation , Monocytes/drug effects , Receptors, Antigen, T-Cell, gamma-delta/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Zoledronic AcidABSTRACT
The permeability estimation from cell monolayer permeation data is usually based on 100% recovery assumption. However, poor recovery is often seen in such experiments in practice but often neglected in data interpretation. In the present study, the cellular retention kinetics during Caco-2 permeation experiments of three passively transported compounds, weakly basic propranolol [(+/-)-1-isopropylamino-3-(1-naphthyloxy)-2-propanol], weakly acidic ibuprofen [alpha-methyl-4-(isobutyl)phenylacetic acid], and neutral testosterone (17beta-hydroxy-4-androsten-3-one), were determined. Furthermore, the effects of cellular retention kinetics on apparent permeability were evaluated, and the role of lysosomal sequestration in cellular retention of propranolol was explored. The cellular retention profiles were observed to be direction and concentration dependent, which may cause erroneous directionality and concentration dependence in permeability estimates. Furthermore, the lysosomal sequestration was demonstrated to contribute to the extent and kinetics of the cellular retention of propranolol.
Subject(s)
Caco-2 Cells/metabolism , Cell Membrane Permeability/physiology , Lysosomes/metabolism , Biological Transport , Cytosol/metabolism , Humans , Ibuprofen/metabolism , Kinetics , Macrolides/metabolism , Macrolides/pharmacology , Models, Biological , Propranolol/metabolism , Testosterone/metabolismABSTRACT
BACKGROUND: Intramedullary implantation causes injury-induced stimulation of intramembranous bone regeneration. Intramedullary bone injury along with stress shielding may induce periimplant bone loss and cause early aseptic loosening of an implant. The aim of this study was to determine the effect of locally administered zoledronic acid on periimplant bone and injury-induced stimulation of intramembranous bone regeneration in a rat model. METHODS: A total of 28 male rats had a titanium implant inserted into their right femur. During the operation, the medullary canal was lavaged using 20 muM zoledronic acid (Zometa 4 mg/5 ml) or sodium chloride. Follow-up times were 4 and 12 weeks, with each follow-up group consisting of seven rats. The femurs with the titanium implants in situ were harvested, and three microscope sections were cut from each femur. The sections were photographed and analyzed with the Analysis computer program. RESULTS: Between 4 and 12 weeks, the length of fluorescence bone contact increased significantly in both groups (control 15.7% SD and zoledronic acid 18.8% SD), although the difference between the groups was not significant. Periimplant bone volume (thickness) was increased in the 4-week zoledronic acid group compared to the controls (+/-13.4%, P = 0.002) but at 12 weeks the groups no longer differed from each other. CONCLUSIONS: Our results suggest that zoledronic acid may prevent injury-induced bone loss near an intramedullary implant by inhibiting bone resorption shortly after implantation. This may provide better periimplant bone stock during the early postoperative period.
Subject(s)
Bone Regeneration/drug effects , Diphosphonates/pharmacology , Femur/surgery , Imidazoles/pharmacology , Osseointegration/drug effects , Titanium , Animals , Biocompatible Materials , Bone Regeneration/physiology , Disease Models, Animal , Femur/drug effects , Fracture Fixation, Intramedullary , Implants, Experimental , Injections, Intralesional , Male , Osseointegration/physiology , Probability , Random Allocation , Rats , Rats, Wistar , Reference Values , Risk Assessment , Zoledronic AcidABSTRACT
The effects of novel quaternary chitosan derivatives on the paracellular transport of mannitol and cell viability were studied in the Caco-2 cell model. The N-betainate derivative with the degree of substitution of 0.05 was very effective at 1.0% (w/v) concentration. The activity decreased as the degree of substitution increased. The cytotoxicity of N-betainates was rather low. The N-piperazines were at least equally effective as the N-betainates with a similar degree of substitution (>0.15). Most of the N-piperazines did not exert toxic effects on the cell monolayers. Overall, the inverse proportionality between the degree of substitution and activity suggests that an intact chitosan backbone is essential for the bioactivity of chitosan derivatives. The quaternary group does not substitute for the activity of the free amine group. In particular, the N-betainate derivatives of chitosan should contain only the minimum number of substituents required for water solubility.
Subject(s)
Betaine/pharmacology , Cell Membrane Permeability/drug effects , Chitosan/pharmacology , Intestinal Mucosa/drug effects , Mannitol/metabolism , Piperazines/pharmacology , Betaine/analogs & derivatives , Betaine/chemistry , Betaine/toxicity , Caco-2 Cells , Cell Survival/drug effects , Chitosan/analogs & derivatives , Chitosan/chemistry , Chitosan/toxicity , Humans , Hydrogen-Ion Concentration , Intestinal Absorption/drug effects , Intestinal Mucosa/metabolism , L-Lactate Dehydrogenase/metabolism , Piperazines/chemistry , Piperazines/toxicity , Solubility , Time FactorsABSTRACT
Caco-2 cells, widely used to study carrier mediated uptake and efflux mechanisms, are known to have different properties when cultured under different conditions. In this study, Caco-2 cells from 10 different laboratories were compared in terms of mRNA expression levels of 72 drug and nutrient transporters, and 17 other target genes, including drug metabolising enzymes, using real-time PCR. The rank order of the top five expressed genes was: HPT1>GLUT3>GLUT5>GST1A>OATP-B. Rank correlation showed that for most of the samples, the gene ranking was not significantly different. Functionality of transporters and the permeability of passive transport markers metoprolol (transcellular) and atenolol (paracellular) were also compared. MDR1 and PepT1 function was investigated using talinolol and Gly-Sar transport, respectively. Sulfobromophthalein (BSP) was used as a marker for MRP2 and OATP-B functionality. Atenolol permeability was more variable across laboratories than metoprolol permeability. Talinolol efflux was observed by all the laboratories, whereas only five laboratories observed significant apical uptake of Gly-Sar. Three laboratories observed significant efflux of BSP. MDR1 expression significantly correlated to the efflux ratio and net active efflux of talinolol. PepT1 mRNA levels showed significant correlation to the uptake ratio and net active uptake of Gly-Sar. MRP2 and OATP-B showed no correlation to BSP transport parameters. Heterogeneity in transporter activity may thus be due to differences in transporter expression as shown for PepT1 and MDR1 which in turn is determined by the culture conditions. Absolute expression of genes was variable indicating that small differences in culture conditions have a significant impact on gene expression, although the overall expression patterns were similar.