ABSTRACT
We have previously shown that C1q is expressed on endothelial cells (ECs) of newly formed decidual tissue. Here we demonstrate that C1q is deposited in wound-healing skin in the absence of C4 and C3 and that C1q mRNA is locally expressed as revealed by real-time PCR and in situ hybridization. C1q was found to induce permeability of the EC monolayer, to stimulate EC proliferation and migration, and to promote tube formation and sprouting of new vessels in a rat aortic ring assay. Using a murine model of wound healing we observed that vessel formation was defective in C1qa(-/-) mice and was restored to normal after local application of C1q. The mean vessel density of wound-healing tissue and the healed wound area were significantly increased in C1q-treated rats. On the basis of these results we suggest that C1q may represent a valuable therapeutic agent that can be used to treat chronic ulcers or other pathological conditions in which angiogenesis is impaired, such as myocardial ischemia.
Subject(s)
Complement C1q/physiology , Endothelial Cells/drug effects , Neovascularization, Physiologic/genetics , Wound Healing/genetics , Animals , Cell Proliferation/drug effects , Complement C1q/genetics , Complement C1q/pharmacology , DNA Primers/genetics , Endothelial Cells/physiology , Enzyme-Linked Immunosorbent Assay , Human Umbilical Vein Endothelial Cells , Humans , Immunoblotting , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Inbred C57BL , Mice, Knockout , Neovascularization, Physiologic/physiology , Rats , Rats, Wistar , Real-Time Polymerase Chain Reaction , Wound Healing/physiologyABSTRACT
Angiogenesis is considered responsible for the growth of primary tumours and of their metastases. With the present study, the effects of three ruthenium compounds, potassiumchlorido (ethylendiamminotetraacetate)rutenate(III) (RuEDTA), sodium (bis-indazole)tetrachloro-ruthenate(III), Na[trans-RuCl4Ind2] (KP1339) and trans-imidazoledimethylsulphoxidetetrachloro-ruthenate (NAMI-A), are studied in vitro in models mimicking the angiogenic process. The ruthenium compounds reduced the production and the release of nitrosyls from either healthy macrophages and immortalized EA.hy926 endothelial cells. The effects of NAMI-A are qualitatively similar and sometimes quantitatively superior to those of RuEDTA and KP1339. NAMI-A reduces the production and release of nitric oxide (NO) by the EA.hy926 endothelial cells and correspondingly inhibits their invasive ability; it also strongly inhibits the angiogenesis in matrigel sponges implanted subcutaneously in healthy mice. Taken together, these data support the anti-angiogenic activity of the tested ruthenium compounds and they contribute to explain the selective activity of NAMI-A against solid tumour metastases, the tumour compartment on which angiogenesis is strongly involved. This anti-angiogenic effect may also contribute to the inhibition of the release of metastatic cells from the primary tumour. Investigations on the anti-angiogenic effects of NAMI-A at this level will increase knowledge of its pharmacological properties and it will give a further impulse to the development of this class of innovative metal-based drugs.
Subject(s)
Coordination Complexes/pharmacology , Free Radical Scavengers/pharmacology , Nitric Oxide/metabolism , Ruthenium/pharmacology , Angiogenesis Inhibitors/pharmacology , Animals , Cell Line , Cell Survival/drug effects , Collagen/pharmacology , Drug Combinations , Humans , Inhibitory Concentration 50 , Laminin/pharmacology , Macrophages, Peritoneal/cytology , Macrophages, Peritoneal/drug effects , Male , Mice , Proteoglycans/pharmacologyABSTRACT
KRAS mutations are primary genetic lesions leading to pancreatic cancer. The promoter of human KRAS contains a nuclease-hypersensitive element (NHE) that can fold in G4-DNA structures binding to nuclear proteins, including MAZ (myc-associated zinc-finger). Here, we report that MAZ activates KRAS transcription. To knockdown oncogenic KRAS in pancreatic cancer cells, we designed oligonucleotides that mimic one of the G-quadruplexes formed by NHE (G4-decoys). To increase their nuclease resistance, two locked nucleic acid (LNA) modifications were introduced at the 3'-end, whereas to enhance the folding and stability, two polycyclic aromatic hydrocarbon units (TINA or AMANY) were inserted internally, to cap the quadruplex. The most active G4-decoy (2998), which had two para-TINAs, strongly suppressed KRAS expression in Panc-1 cells. It also repressed their metabolic activity (IC50 = 520 nM), and it inhibited cell growth and colony formation by activating apoptosis. We finally injected 2998 and control oligonucleotides 5153, 5154 (2 nmol/mouse) intratumorally in SCID mice bearing a Panc-1 xenograft. After three treatments, 2998 reduced tumor xenograft growth by 64% compared with control and increased the Kaplan-Meier median survival time by 70%. Together, our data show that MAZ-specific G4-decoys mimicking a KRAS quadruplex are promising for pancreatic cancer therapy.
Subject(s)
Antineoplastic Agents/therapeutic use , DNA-Binding Proteins/metabolism , Pancreatic Neoplasms/drug therapy , Proto-Oncogene Proteins/antagonists & inhibitors , Transcription Factors/metabolism , ras Proteins/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis , Binding Sites , Binding, Competitive , Cell Line, Tumor , DNA/chemistry , G-Quadruplexes , Humans , Mice , Mice, SCID , Oligonucleotides/chemistry , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Promoter Regions, Genetic , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins p21(ras) , Transcriptional Activation , ras Proteins/genetics , ras Proteins/metabolismABSTRACT
BACKGROUND: Porphyrin TMPyP4 (P4) and its C14H28-alkyl derivative (C14) are G-quadruplex binders and singlet oxygen (1O2) generators. In contrast, TMPyP2 (P2) produces 1O2 but it is not a G-quadruplex binder. As their photosensitizing activity is currently undefined, we report in this study their efficacy against a melanoma skin tumour and describe an in vitro mechanistic study which gives insights into their anticancer activity. METHODS: Uptake and antiproliferative activity of photoactivated P2, P4 and C14 have been investigated in murine melanoma B78-H1 cells by FACS, clonogenic and migration assays. Apoptosis was investigated by PARP-1 cleavage and annexin-propidium iodide assays. Biodistribution and in vivo anticancer activity were tested in melanoma tumour-bearing mice. Porphyrin binding and photocleavage of G-rich mRNA regions were investigated by electrophoresis and RT-PCR. Porphyrin effect on ERK pathway was explored by Western blots. RESULTS: Thanks to its higher lipophylicity C14 was taken up by murine melanoma B78-H1 cells up to 30-fold more efficiently than P4. When photoactivated (7.2 J/cm2) in B78-H1 melanoma cells, P4 and C14, but not control P2, caused a strong inhibition of metabolic activity, clonogenic growth and cell migration. Biodistribution studies on melanoma tumour-bearing mice showed that P4 and C14 localize in the tumour. Upon irradiation (660 nm, 193 J/cm2), P4 and C14 retarded tumour growth and increased the median survival time of the treated mice by ~50% (P <0.01 by ANOVA), whereas porphyrin P2 did not. The light-dependent mechanism mediated by P4 and C14 is likely due to the binding to and photocleavage of G-rich quadruplex-forming sequences within the 5'-untranslated regions of the mitogenic ras genes. This causes a decrease of RAS protein and inhibition of downstream ERK pathway, which stimulates proliferation. Annexin V/propidium iodide and PARP-1 cleavage assays showed that the porphyrins arrested tumour growth by apoptosis and necrosis. C14 also showed an intrinsic light-independent anticancer activity, as recently reported for G4-RNA binders. CONCLUSIONS: Porphyrins P4 and C14 impair the clonogenic growth and migration of B78-H1 melanoma cells and inhibit melanoma tumour growth in vivo. Evidence is provided that C14 acts through light-dependent (mRNA photocleavage) and light-independent (translation inhibition) mechanisms.
Subject(s)
Photochemotherapy , Photosensitizing Agents/administration & dosage , Porphyrins/administration & dosage , Skin Neoplasms/drug therapy , Animals , Apoptosis/drug effects , Cell Line, Tumor , G-Quadruplexes/drug effects , Humans , MAP Kinase Signaling System/drug effects , Mice , Porphyrins/chemistryABSTRACT
Pheophorbide a (Pba) is a chlorophyll catabolite that has been proposed as photosensitizer in photodynamic therapy. In a previous study we conjugated Pba to monomethoxy-polyethylene glycol (mPEG-Pba), to increase its solubility and pharmacokinetics. Here, we compare the photodynamic therapy efficacy of free Pba and mPEG-Pba to cure a subcutaneous amelanotic melanoma transplanted in C57/BL6 mice. The photosensitizers, i.p. injected (30 mg/kg), showed no toxicity when the animals were kept in the dark. But, after photoactivation with a 660 nm laser (fluence of 193 J/cm(2)), both photosensitizers, in particular mPEG-Pba, showed a strong efficacy to cure the tumor, both in terms of tumor growth delay and increase of Kaplan-Meier median survival time. Together, our in vivo data demonstrate that mPEG-conjugated Pba is a promising photosensitizer for the photodynamic therapy of cancer.
Subject(s)
Chlorophyll/analogs & derivatives , Melanoma, Amelanotic/drug therapy , Polyethylene Glycols/chemistry , Radiation-Sensitizing Agents/administration & dosage , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Chlorophyll/administration & dosage , Chlorophyll/chemistry , Female , Light , Mice , Mice, Inbred C57BL , Photochemotherapy , Radiation-Sensitizing Agents/chemistryABSTRACT
Cell recurrence in cancer photodynamic therapy (PDT) is an important issue that is poorly understood. It is becoming clear that nitric oxide (NO) is a modulator of PDT. By acting on the NF-κB/Snail/RKIP survival/anti-apoptotic loop, NO can either stimulate or inhibit apoptosis. We found that pheophorbide a/PDT (Pba/PDT) induces the release of NO in B78-H1 murine amelanotic melanoma cells in a concentration-dependent manner. Low-dose PDT induces low NO levels by stimulating the anti-apoptotic nature of the above loop, whereas high-dose PDT stimulates high NO levels inhibiting the loop and activating apoptosis. When B78-H1 cells are treated with low-dose Pba/PDT and DETA/NO, an NO-donor, intracellular NO increases and cell growth is inhibited according to scratch-wound and clonogenic assays. Western blot analyses showed that the combined treatment reduces the expression of the anti-apoptotic NF-κB and Snail gene products and increases the expression of the pro-apoptotic RKIP gene product. The combined effect of Pba and DETA/NO was also tested in C57BL/6 mice bearing a syngeneic B78-H1 melanoma. We used pegylated Pba (mPEG-Pba) due to its better pharmacokinetics compared to free Pba. mPEG-Pba (30 mg/Kg) and DETA/NO (0.4 mg/Kg) were i.p. injected either as a single molecule or in combination. After photoactivation at 660 nM (fluence of 193 J/cm(2)), the combined treatment delays tumor growth more efficiently than each individual treatment (p<0.05). Taken together, our results showed that the efficacy of PDT is strengthened when the photosensitizer is used in combination with an NO donor.
Subject(s)
Chlorophyll/analogs & derivatives , Melanoma, Amelanotic/drug therapy , Nitric Oxide Donors/pharmacology , Nitric Oxide/metabolism , Photochemotherapy/methods , Photosensitizing Agents/pharmacology , Skin Neoplasms/drug therapy , Animals , Cell Line, Tumor , Chlorophyll/pharmacology , Female , Flow Cytometry , Melanoma, Amelanotic/metabolism , Mice , Mice, Inbred C57BL , NG-Nitroarginine Methyl Ester/pharmacology , Nitroso Compounds/pharmacology , Skin Neoplasms/metabolism , Wound Healing/drug effectsABSTRACT
A new single-chain fragment variable (scFv) to TRAIL-R2 receptor produced as minibody (MB2.23) was characterized for anti-lymphoma activity in vivo. For this purpose, a disseminated lymphoma model was generated by intraperitoneal inoculation of BJAB cells in severe combined immunodeficiency mice. Two weekly injections with MB2.23 (10 mg/kg) were able to significantly increase the median survival time of lymphoma-bearing animals with respect to the vehicle-treated control mice, providing a rationale for further investigating the use of MB2.23 in anticancer therapy.
Subject(s)
Immunotherapy/methods , Lymphoma, B-Cell/therapy , Receptors, TNF-Related Apoptosis-Inducing Ligand/immunology , Single-Chain Antibodies/administration & dosage , Animals , Humans , Injections, Intraperitoneal , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/pathology , Mice , Mice, SCID , Time Factors , Xenograft Model Antitumor AssaysABSTRACT
Exploiting the tumor environment features (EPR effect, elevated glutathione, reactive oxygen species levels) might allow attaining a selective and responsive carrier capable of improving the therapeutic outcome. To this purpose, the in situ covalent binding of drugs and nanoparticles to circulating human serum albumin (HSA) might represent a pioneering approach to achieve an effective strategy. This study describes the synthesis, in vitro and in vivo evaluation of bioresponsive HSA-binding nanoparticles (MAL-PTX2S@Pba), co-delivering two different paclitaxel (PTX) prodrugs and the photosensitizer pheophorbide a (Pba), for the combined photo- and chemo-treatment of breast cancer. Stable and reproducible MAL-PTX2S@Pba nanoparticles with an average diameter of 82 nm and a PTX/Pba molar ratio of 2.5 were obtained by nanoprecipitation. The in vitro 2D combination experiments revealed that MAL-PTX2S@Pba treatment induces a strong inhibition of cell viability of MDA-MB-231, MCF7 and 4T1 cell lines, whereas 3D experiments displayed different trends: while MAL-PTX2S@Pba effectiveness was confirmed against MDA-MB-231 spheroids, the 4T1 model exhibited marked resistance. Lastly, despite using a low PTX-PDT regimen (e.g., 8.16 mg/Kg PTX and 2.34 mg/Kg Pba), our formulation showed to foster primary tumor reduction and curb lung metastases growth in 4T1 tumor-bearing mice, thus setting the basis for further preclinical validations.
ABSTRACT
The complement system is a major component of humoral innate immunity, acting as a first line of defense against microbes via opsonization and lysis of pathogens. However, novel roles of the complement system in inflammatory and immunological processes, including in cancer, are emerging. Endometriosis (EM), a benign disease characterized by ectopic endometrial implants, shows certain unique features of cancer, such as the capacity to invade surrounding tissues, and in severe cases, metastatic properties. A defective immune surveillance against autologous tissue deposited in the peritoneal cavity allows immune escape for endometriotic lesions. There is evidence that the glandular epithelial cells found in endometriotic implants produce and secrete the complement component C3. Here, we show, using immunofluorescence and RT-qPCR, the presence of locally synthesized C3 in the ectopic endometriotic tissue, but not in the eutopic tissue. We generated a murine model of EM via injection of minced uterine tissue from a donor mouse into the peritoneum of recipient mice. The wild type mice showed greater amount of cyst formation in the peritoneum compared to C3 knock-out mice. Peritoneal washings from the wild type mice with EM showed more degranulated mast cells compared to C3 knock-out mice, consistent with higher C3a levels in the peritoneal fluid of EM patients. We provide evidence that C3a participates in an auto-amplifying loop leading to mast cell infiltration and activation, which is pathogenic in EM. Thus, C3 can be considered a marker of EM and its local synthesis can promote the engraftment of the endometriotic cysts.
Subject(s)
Cell Degranulation , Complement C3/metabolism , Endometriosis/metabolism , Endometrium/metabolism , Inflammation Mediators/metabolism , Mast Cells/metabolism , Peritoneal Diseases/metabolism , Animals , Case-Control Studies , Coculture Techniques , Complement C3/genetics , Complement C3a/metabolism , Disease Models, Animal , Endometriosis/genetics , Endometriosis/immunology , Endometrium/drug effects , Endometrium/immunology , Endometrium/transplantation , Female , Hep G2 Cells , Humans , Immunity, Humoral , Immunity, Innate , Mast Cells/immunology , Mice, Inbred C57BL , Mice, Knockout , Peritoneal Diseases/genetics , Peritoneal Diseases/immunology , Signal Transduction , THP-1 Cells , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
BACKGROUND: Bac7 is a proline-rich peptide with a potent in vitro antimicrobial activity against Gram-negative bacteria. Here we investigated its activity in biological fluids and in vivo using a mouse model of S. typhimurium infection. RESULTS: The efficacy of the active 1-35 fragment of Bac7 was assayed in serum and plasma, and its stability in biological fluids analyzed by Western blot and mass spectrometry. The ability of the peptide to protect mice against Salmonella was assayed in a typhoid fever model of infection by determination of survival rates and bacterial load in liver and spleen of infected animals. In addition, the peptide's biodistribution was evaluated by using time-domain optical imaging. Bac7(1-35) retained a substantial in vivo activity showing a very low toxicity. The peptide increased significantly the number of survivors and the mean survival times of treated mice reducing the bacterial load in their organs despite its rapid clearance. CONCLUSIONS: Our results provide a first indication for a potential development of Bac7-based drugs in the treatment of salmonellosis and, eventually, other Gram-negative infections. The in vivo activity for this peptide might be substantially enhanced by decreasing its excretion rate or modifying the treatment schedule.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Salmonella Infections, Animal/drug therapy , Salmonella typhimurium , Animals , Antimicrobial Cationic Peptides/pharmacokinetics , Liver/microbiology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Salmonella Infections, Animal/microbiology , Spleen/microbiology , Tissue DistributionABSTRACT
Designing small molecules able to break down G4 structures in mRNA (RG4s) offers an interesting approach to cancer therapy. Here, we have studied cationic porphyrins (CPs) bearing an alkyl chain up to 12 carbons, as they bind to RG4s while generating reactive oxygen species upon photoirradiation. Fluorescence-activated cell sorting (FACS) and confocal microscopy showed that the designed alkyl CPs strongly penetrate cell membranes, binding to KRAS and NRAS mRNAs under low-abundance cell conditions. In Panc-1 cells, alkyl CPs at nanomolar concentrations promote a dramatic downregulation of KRAS and NRAS expression, but only if photoactivated. Alkyl CPs also reduce the metabolic activity of pancreatic cancer cells and the growth of a Panc-1 xenograft in SCID mice. Propidium iodide/annexin assays and caspase 3, caspase 7, and PARP-1 analyses show that these compounds activate apoptosis. All these data demonstrate that the designed alkyl CPs are efficient photosensitizers for the photodynamic therapy of ras-driven cancers.
Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Pancreatic Ductal/drug therapy , G-Quadruplexes/drug effects , Pancreatic Neoplasms/drug therapy , Photosensitizing Agents/therapeutic use , Porphyrins/therapeutic use , Animals , Antineoplastic Agents/chemical synthesis , Apoptosis/drug effects , Base Sequence , Cell Line, Tumor , Down-Regulation/drug effects , Female , GTP Phosphohydrolases/genetics , Genes, ras/drug effects , Humans , Membrane Proteins/genetics , Mice, SCID , Photochemotherapy/methods , Photosensitizing Agents/chemical synthesis , Porphyrins/chemical synthesis , Proto-Oncogene Proteins p21(ras)/genetics , RNA/chemistry , RNA/genetics , Reactive Oxygen Species/metabolismABSTRACT
BACKGROUND: The effectiveness of therapies for chronic lymphocytic leukemia (CLL), the most common leukemia in Western countries adults, can be improved via a deeper understanding of its molecular abnormalities. Whereas the isoforms of the eukaryotic elongation factor 1A (eEF1A1 and eEF1A2) are implicated in different tumors, no information are available in CLL. METHODS: eEF1A1/eEF1A2 amounts were quantitated in the lymphocytes of 46 CLL patients vs normal control (real time PCR, western blotting). eEF1A1 role in CLL was investigated in a cellular (MEC-1) and animal model of CLL via its targeting by an aptamer (GT75) or a siRNA (siA1) delivered by electroporation (in vitro) or lipofection (in vivo). RESULTS: eEF1A1/eEF1A2 were elevated in CLL lymphocytes vs control. eEF1A1 but not eEF1A2 levels were higher in patients which died during the study compared to those surviving. eEF1A1 targeting (GT75/siA1) resulted in MEC-1 viability reduction/autophagy stimulation and in vivo tumor growth down-regulation. CONCLUSIONS: The increase of eEF1A1 in dead vs surviving patients may confer to eEF1A1 the role of a prognostic marker for CLL and possibly of a therapeutic target, given its involvement in MEC-1 survival. Specific aptamer/siRNA released by optimized delivery systems may allow the development of novel therapeutic options.
Subject(s)
Aptamers, Nucleotide/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Peptide Elongation Factor 1/genetics , RNA, Small Interfering/genetics , Aged , Animals , Cell Line, Tumor , Cell Proliferation/genetics , Down-Regulation/genetics , Female , Humans , Male , Mice , Mice, SCID , Protein Isoforms/geneticsABSTRACT
Complement C1q is the activator of the classical pathway. However, it is now recognized that C1q can exert functions unrelated to complement activation. Here we show that C1q, but not C4, is expressed in the stroma and vascular endothelium of several human malignant tumours. Compared with wild-type (WT) or C3- or C5-deficient mice, C1q-deficient (C1qa(-/-)) mice bearing a syngeneic B16 melanoma exhibit a slower tumour growth and prolonged survival. This effect is not attributable to differences in the tumour-infiltrating immune cells. Tumours developing in WT mice display early deposition of C1q, higher vascular density and an increase in the number of lung metastases compared with C1qa(-/-) mice. Bone marrow (BM) chimeras between C1qa(-/-) and WT mice identify non-BM-derived cells as the main local source of C1q that can promote cancer cell adhesion, migration and proliferation. Together these findings support a role for locally synthesized C1q in promoting tumour growth.
Subject(s)
Complement Activation/physiology , Complement C1q/metabolism , Neoplasms/metabolism , Animals , Apoptosis , Cell Line, Tumor , Cell Movement , Cell Proliferation , Complement C1q/genetics , Complement C3/genetics , Complement C3/metabolism , Complement C5/genetics , Complement C5/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
PURPOSE: The purpose is to evaluate the CD44-mediated cellular targeting of HA-But, a hyaluronic acid esterified with butyric acid (But) residues, to hepatocellular carcinoma cell lines in vitro and to hepatic tumor metastases in vivo. EXPERIMENTAL DESIGN: In vitro, the CD44-dependent cytotoxicity in two human hepatocellular carcinoma cell lines (HepB3 and HepG2) with high and low CD44 expression was investigated; in vivo, the effect on liver metastases originating from intrasplenic implants of Lewis lung carcinoma (LL3) or B16-F10 melanoma in mice was compared with the pharmacokinetics of organ and tissue distribution using different routes of administration. RESULTS: HepB3 and HepG2 cell lines showed different expression of CD44 (78 and 18%, respectively), which resulted in a CD44-dependent HA-But inhibitory effect as demonstrated also by the uptake analysis performed using radiolabeled HA-But ((99m)Tc-HA-But). Pharmacokinetic studies showed different rates of (99m)Tc-HA-But distribution according to the route of administration (i.v., i.p., or s.c.): very fast (a few minutes) after i.v. treatment, with substantial accumulation in the liver and spleen; relatively slow after i.p. or s.c. treatment, with marked persistence of the drug at the site of injection. The effect of s.c. and i.p. treatment with HA-But on liver metastases originating from intrasplenic implants of LL3 carcinoma or B16-F10 melanoma (both CD44-positive: 68 and 87%, respectively), resulted in 87 and 100% metastases-free animals, respectively (regardless of the route of administration), and a significant prolongation of the life expectancy compared with control groups. CONCLUSIONS: HA-But tends to concentrate in the liver and spleen and appears to be a promising new drug for the treatment of intrahepatic tumor lesions.
Subject(s)
Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Liver Neoplasms/prevention & control , Animals , Butyric Acid/chemistry , Butyric Acid/pharmacokinetics , Butyric Acid/pharmacology , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/prevention & control , Carcinoma, Lewis Lung/metabolism , Carcinoma, Lewis Lung/pathology , Carcinoma, Lewis Lung/prevention & control , Cell Line, Tumor , Cell Survival/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Esters , Female , Flow Cytometry , Humans , Hyaluronan Receptors/analysis , Hyaluronic Acid/chemistry , Hyaluronic Acid/pharmacokinetics , Hyaluronic Acid/pharmacology , Liver Neoplasms/pathology , Liver Neoplasms/secondary , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Melanoma, Experimental/prevention & control , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Inbred DBA , Neoplasm Metastasis/pathology , Neoplasm Metastasis/prevention & control , Organotechnetium Compounds/chemistry , Organotechnetium Compounds/pharmacokinetics , Survival Analysis , Tissue DistributionABSTRACT
NAMI-A is a ruthenium complex endowed with a selective effect on lung metastases of solid metastasizing tumors. The aim of this study is to provide evidence that NAMI-A's effect is based on the selective sensitivity of the metastasis cell, as compared with other tumor cells, and to show that lungs represent a privileged site for the antimetastatic effects. The transplantation of Lewis lung carcinoma cells, harvested from the primary tumor of mice treated with 35 mg/kg/day NAMI-A for six consecutive days, a dose active on metastases, shows no change in primary tumor take and growth but a significant reduction in formation of spontaneous lung metastases. Transmission electron microscopy examination of lungs and kidney shows NAMI-A to selectively bind collagen of the lung extracellular matrix and also type IV collagen of the basement membrane of kidney glomeruli. The half lifetime of NAMI-A elimination from the lungs is longer than for liver, kidney, and primary tumor. NAMI-A bound to collagen is active on tumor cells as shown in vitro by an invasion test, using a modified Boyden chamber and Matrigel, and it inhibits the matrix metallo-proteinases MMP-2 and MMP-9 at micromolar concentrations, as shown in vitro by a zimography test. These data show NAMI-A to significantly affect tumor cells with metastatic ability. Binding to collagen allows NAMI-A to exert its selective activity on metastatic cells during dissemination and particularly in the lungs. These data also stress the wide spectrum of daily doses and treatment schedules at which NAMI-A is active against metastases.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Lewis Lung/prevention & control , Collagen Type IV/metabolism , Dimethyl Sulfoxide/analogs & derivatives , Dimethyl Sulfoxide/metabolism , Dimethyl Sulfoxide/pharmacology , Lung Neoplasms/prevention & control , Mammary Neoplasms, Experimental/prevention & control , Organometallic Compounds/metabolism , Organometallic Compounds/pharmacology , Adenocarcinoma/prevention & control , Adenocarcinoma/secondary , Animals , Carcinoma, Lewis Lung/secondary , Cell Adhesion , Cell Division/drug effects , Collagen , Drug Combinations , Female , Humans , Kidney/ultrastructure , Laminin , Lung/ultrastructure , Lung Neoplasms/pathology , Mammary Neoplasms, Experimental/secondary , Matrix Metalloproteinase Inhibitors , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Neoplasm Invasiveness/prevention & control , Proteoglycans , Ruthenium/metabolism , Ruthenium Compounds , Tumor Cells, CulturedABSTRACT
We previously found that two neighboring G-quadruplexes behave as a molecular switch controlling the expression of HRAS (Cogoi, S.; Schekotikhin, A. E.; Xodo, L. E. Nucl. Acids Res. 2014, DOI: 10.1093/nar/gku574). In this study we have designed anthrathiophenediones with two chloroacetamidine-containing side chains (CATDs) as G-quadruplex binders and have examined their anticancer activity in T24 bladder cancer cells bearing mutant HRAS and in T24 xenografts. The designed CATDs (3a-e), bearing alkyl side chains of different length, penetrate T24 cancer cells more than their analogues with guanidine-containing side chains. The lead compounds 3a and 3c inhibit HRAS expression, metabolic activity, and colony formation in T24 cancer cells. They also activate a strong apoptotic response, as indicated by PARP-1, caspases 3/7, and annexin V/propidium iodide assays. Apoptosis occurs under conditions where cyclin D1 is down-regulated and the cell cycle arrested in G2 phase. Finally, compound 3a inhibits the growth of T24 xenografts and increases the median survival time of nude mice.
Subject(s)
Acetamides/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Thiophenes/chemistry , Thiophenes/pharmacology , Urinary Bladder Neoplasms/pathology , Alleles , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Biological Transport , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin D1/metabolism , Drug Design , G-Quadruplexes/drug effects , G2 Phase Cell Cycle Checkpoints/drug effects , Genes, ras/genetics , Humans , Mice , Substrate Specificity , Survival Analysis , Thiophenes/chemical synthesis , Thiophenes/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Two fullerene derivatives (fullerenes 1 and 2), bearing a hydrophilic chain on the pyrrolidinic nitrogen, were developed with the aim to deliver anticancer agents to solid tumors. These two compounds showed a significantly different behaviour on human neoplastic cell lines in vitro in respect to healthy leukocytes. In particular, the pyrrolidinium ring on the fullerene carbon cage brings to a more active compound. In the present work, we describe the effects of these fullerenes on primary cultures of human monocytes and macrophages, two kinds of immune cells representing the first line of defence in the immune response to foreign materials. These compounds are not recognized by circulating monocytes while they get into macrophages. The evaluation of the pronecrotic or proapoptotic effects, analysed by means of analysis of the purinergic receptor P2X7 activation and of ROS scavenging activity, has allowed us to show that fullerene 2, but not its analogue fullerene 1, displays toxicity, even though at concentrations higher than those shown to be active on neoplastic cells.
Subject(s)
Antineoplastic Agents/administration & dosage , Fullerenes/administration & dosage , Macrophages/drug effects , Neoplasms/drug therapy , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Cell Line, Tumor , Fullerenes/chemistry , Humans , Hydrophobic and Hydrophilic Interactions , Monocytes/drug effects , Neoplasms/pathology , Nitrogen/chemistry , Receptors, Purinergic P2X7/biosynthesis , Receptors, Purinergic P2X7/geneticsABSTRACT
The expectations of nanoparticle (NP)-based targeted drug delivery systems in cancer, when compared with convectional therapeutic methods, are greater efficacy and reduced drug side effects due to specific cellular-level interactions. However, there are conflicting literature reports on enhanced tumor accumulation of targeted NPs, which is essential for translating their applications as improved drug-delivery systems and contrast agents in cancer imaging. In this study, we characterized biodegradable NPs conjugated with an anti-CD20 antibody for in vivo imaging and drug delivery onto tumor cells. NPs' binding specificity mediated by anti-CD20 antibody was evaluated on MEC1 cells and chronic lymphocytic leukemia patients' cells. The whole-body distribution of untargeted NPs and anti-CD20 NPs were compared by time-domain optical imaging in a localized human/mouse model of B-cell malignancy. These studies provided evidence that NPs' functionalization by an anti-CD20 antibody improves tumor pharmacokinetic profiles in vivo after systemic administration and increases in vivo imaging of tumor mass compared to non-targeted NPs. Together, drug delivery and imaging probe represents a promising theranostics tool for targeting B-cell malignancies.
Subject(s)
Antigens, CD20/chemistry , Drug Delivery Systems , Leukemia, B-Cell/diagnosis , Nanoparticles/chemistry , Polymers/chemistry , Animals , Cell Line, Tumor , Humans , Mice , Molecular ImagingABSTRACT
Ruthenium compounds have gained large interest for their potential application as chemotherapeutic agents, and in particular the complexes of the type (X)[trans-RuCl4(dmso-S)L] (X = HL or Na, NAMI-A or NAMI, respectively, for L = imidazole) are under investigation for their antimetastatic properties. The NAMI(-A)-like compounds are prodrugs that hydrolyze in vivo, and the investigation of their hydrolytic properties is therefore important for determining the nature of the potential active species. The NAMI-A-type Ru(III) complex 1, (Hdmtp)[trans-RuCl4(dmso-S)(dmtp)] (dmtp is 5,7-dimethyl[1,2,4]triazolo[1,5-a]pyrimidine), and the corresponding sodium analogue 2, (Na)[trans-RuCl4(dmso-S)(dmtp)], were synthesized. The hydrolyses of 1 and 2 in water as well as in buffered solutions were studied, and the first hydrolysis product, [mer-RuCl3(H2O)(dmso-S)(dmtp)].H2O (3), was isolated and characterized. The molecular structures of 1 and 3 were determined by single-crystal X-ray diffraction analyses and prove the importance of the hydrogen-bonding properties of dmtp to stabilize hydrolysis products. In vitro 1 (a) is not cytotoxic on tumor cells, following challenges from 1 to 72 h and concentrations up to 100 microM, (b) inhibits matrigel invasion at 0.1 mM and MMP-9 activity with an IC50 of about 1 mM, and (c) is devoid of pronounced effects on cell distribution among cell cycle phases. In vivo compound 1, similar to NAMI-A, significantly inhibits metastasis growth in mice bearing advanced MCa mammary carcinoma tumors. In the lungs, 1 is significantly less concentrated than NAMI-A, whereas no differences between these two compounds were found in other organs such as tumor, liver, and kidney. However, 1 caused edema and necrotic areas on liver parenchyma that are more pronounced than those caused by NAMI-A. Conversely, glomerular and tubular changes on kidney are less extensive than with NAMI-A. In conclusion, 1 confirms the excellent antimetastatic properties of this class of NAMI-A-type compounds and qualifies as an interesting alternative to NAMI-A for treating human cancers.
Subject(s)
Antineoplastic Agents/chemical synthesis , Neoplasm Metastasis/prevention & control , Organometallic Compounds/chemical synthesis , Ruthenium , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Crystallography, X-Ray , Drug Screening Assays, Antitumor , Hydrolysis , Kidney/drug effects , Kidney/pathology , Liver/drug effects , Liver/pathology , Lung Neoplasms/drug therapy , Lung Neoplasms/secondary , Magnetic Resonance Spectroscopy , Mammary Neoplasms, Animal/pathology , Matrix Metalloproteinase 9/chemistry , Mice , Molecular Structure , Neoplasm Invasiveness , Organometallic Compounds/chemistry , Organometallic Compounds/pharmacology , Ruthenium/pharmacokinetics , Spectrophotometry, Ultraviolet , Structure-Activity Relationship , Tissue DistributionABSTRACT
A series of analogues of NAMI-A, a reference compound active on solid tumor metastases, were synthesized (NAMI-A type complexes). They share the same chemical structure of NAMI-A, and differ from it in the nature of the coordinated nitrogen ligand, such as pyrazole, thiazole and pyrazine, which are less basic than imidazole. This modification confers to the new NAMI-A type complexes a better stability in aqueous solution compared to the parent compound, a very important characteristic for a class of compounds that, with NAMI-A, is currently completing a phase I clinical trial at the Netherlands Cancer Institute of Amsterdam. Cytotoxicity and the effects on cell cycle and invasion were investigated on TS/A, B16-F10 and MCF-7 tumor cell lines, while the inhibition of lung metastases was determined on the mouse experimental tumors Lewis lung carcinoma and MCa mammary carcinoma. The new complexes show a pharmacological activity very similar to that of the parental compound NAMI-A: in vitro they are devoid of meaningful cytotoxicity against tumor cells, and in vivo they inhibit metastasis formation and growth approximately to the same extent as NAMI-A. Thus the new NAMI-A type complexes retain the same potent characteristic of NAMI-A to selectively interact with solid tumor metastases. However, compared to NAMI-A they do not stop cell cycle progression at G2-M level and are more active in preventing the spontaneous invasion of Matrigel by tumor cells exposed for 1 h to 10(-4) M concentration. Globally, these complexes take advantage of the knowledge on NAMI-A and appear particularly interesting for future clinical handling and applications.