ABSTRACT
Metabolic adaptations are central for carcinogenesis and response to therapy, but little is known about the contribution of mitochondrial dynamics to the response of glioma cells to the standard treatment with temozolomide (TMZ). Glioma cells responded to TMZ with mitochondrial mass increased and the production of round structures of dysfunctional mitochondria. At single-cell level, asymmetric mitosis contributed to the heterogeneity of mitochondrial levels. It affected the fitness of cells in control and treated condition, indicating that the mitochondrial levels are relevant for glioma cell fitness in the presence of TMZ.
Subject(s)
Brain Neoplasms , Glioma , Humans , Temozolomide/pharmacology , Temozolomide/therapeutic use , Dacarbazine/pharmacology , Dacarbazine/metabolism , Dacarbazine/therapeutic use , Apoptosis , Cell Line, Tumor , Glioma/drug therapy , Glioma/metabolism , Mitochondria/metabolism , Antineoplastic Agents, Alkylating/pharmacology , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Drug Resistance, NeoplasmABSTRACT
Dacarbazine (DTIC) is a chemotherapeutic drug currently used for the systemic treatment of melanomas. Considering the easy access to these tumors, a topical route of drug administration could provide a more comfortable and less toxic treatment. However, DTIC quantification aiming at the design of topical formulations is challenging, pondering all the interferents present in the drug samples recovered from the skin. Hence, this work intended to validate a selective chromatographic method for DTIC determination in skin permeation studies. A reversed-phase C18 column was used as a stationary phase, and gradient elution of a mobile phase consisting of methanol and pH 6.5 sodium phosphate monohydrate buffer (0.01 mol/L) at a flow rate of 1.0 mL/min was implemented. DTIC was detected at 364 nm. The method was selective against skin interferents, linear (r = 0.9995) in a concentration range of 1.0-15.0 µg/mL, precise with an overall variation coefficient lower than 3.8%, accurate achieving recovery from the skin layers within 91-112%, and sensitive for the proposed application (detection limit = 0.10 µg/ mL, quantification limit = 0.30 µg/mL). Furthermore, the analytical method was successfully tested in in vitro skin permeation studies. In conclusion, the developed method is appropriate for DTIC analysis from the skin sample matrix.
Subject(s)
Dacarbazine , Melanoma , Humans , Dacarbazine/analysis , Dacarbazine/metabolism , Skin/metabolism , Skin Absorption , Chromatography, High Pressure Liquid/methodsABSTRACT
Melanoma is a highly metastatic and rapidly progressing cancer, a leading cause of mortality among skin cancers. The melanoma microenvironment, formed from the activity of malignant cells on the extracellular matrix and the recruitment of immune cells, plays an active role in the development of drug resistance and tumor recurrence, which are clinical challenges in cancer treatment. These tumoral metabolic processes are affected by proteins, including Galectin-3 (Gal-3), which is extensively involved in cancer development. Previously, we characterized a partially methylated mannogalactan (MG-Pe) with antimelanoma activities. In vivo models of melanoma were used to observe MG-Pe effects in survival, spontaneous, and experimental metastases and in tissue oxidative stress. Analytical assays for the molecular interaction of MG-Pe and Gal-3 were performed using a quartz crystal microbalance, atomic force microscopy, and contact angle tensiometer. MG-Pe exhibits an additive effect when administered together with the chemotherapeutic agent dacarbazine, leading to increased survival of treated mice, metastases reduction, and the modulation of oxidative stress. MG-Pe binds to galectin-3. Furthermore, MG-Pe antitumor effects were substantially reduced in Gal-3/KO mice. Our results showed that the novel Gal-3 ligand, MG-Pe, has both antitumor and antimetastatic effects, alone or in combination with chemotherapy.
Subject(s)
Antineoplastic Agents , Galectin 3 , Melanoma , Skin Neoplasms , Animals , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Dacarbazine/metabolism , Dacarbazine/pharmacology , Dacarbazine/therapeutic use , Galectin 3/metabolism , Galectin 3/pharmacology , Galectin 3/therapeutic use , Ligands , Melanoma/drug therapy , Melanoma/metabolism , Mice , Neoplasm Recurrence, Local , Skin Neoplasms/drug therapy , Skin Neoplasms/metabolism , Tumor Microenvironment/drug effects , Tumor Microenvironment/physiologyABSTRACT
The prognostic impact of the presence of Epstein-Barr virus (EBV) in classical Hodgkin lymphoma (cHL) is controversial. Previous studies reported heterogeneous results, rendering difficult the clinical validation of EBV as a prognostic biomarker in this lymphoma. The objective of this study was to evaluate the survival impact of the expression of EBV Latent-Membrane Protein 1 (EBV-LMP1) in tumoral Hodgkin-Reed-Sternberg (HRS) cells of primary diagnostic samples of cHL. Formalin-Fixed Paraffin-Embedded (FFPE) lymph node samples from 88 patients with cHL were analyzed. Patients were treated with the standard first-line chemotherapy (CT) with Adriamycin, Bleomycin, Vinblastine and Dacarbazine (ABVD) followed by radiotherapy. The Kaplan-Meier method and the Cox proportional hazards model were used for carrying out the survival analysis. In order to investigate whether the influence of EBV was age-dependent, analyses were performed both for patients of all ages and for age-stratified subgroups. In bivariate analysis, the expression of EBV was associated with older age (p = 0.011), mixed cellularity subtype cHL (p < 0.001) and high risk International Prognostic Score (IPS) (p = 0.023). Overall survival (OS) and progression-free survival (PFS) were associated with the presence of bulky disease (p = 0.009) and advanced disease at diagnosis (p = 0.016). EBV-positive cases did not present a significantly lower OS and PFS in comparison with EBV-negative cases, for all ages and when stratifying for age. When adjusted for covariates, absence of bulky disease at diagnosis (HR: 0.102, 95% CI: 0.02-0.48, p = 0.004) and limited disease stages (I-II) (HR: 0.074, 95% CI: 0.01-0.47, p = 0.006) were associated with a significant better OS. For PFS, limited-disease stages also retained prognostic impact in the multivariate Cox regression (HR: 0.145, 95% CI: 0.04-0.57, p = 0.006). These results are of importance as the early identification of prognostic biomarkers in cHL is critical for guiding and personalizing therapeutic decisions. The prognostic role of EBV in cHL could be modulated by the type of CT protocol employed and interact with the rest of presenting features.
Subject(s)
Epstein-Barr Virus Infections/metabolism , Epstein-Barr Virus Infections/virology , Herpesvirus 4, Human , Hodgkin Disease/metabolism , Hodgkin Disease/virology , Membrane Proteins/metabolism , Adult , Aged , Aged, 80 and over , Bleomycin/pharmacology , Dacarbazine/metabolism , Doxorubicin/pharmacology , Epstein-Barr Virus Infections/pathology , Female , Herpesvirus 4, Human/drug effects , Hodgkin Disease/drug therapy , Hodgkin Disease/pathology , Humans , Male , Middle Aged , Prognosis , Reed-Sternberg Cells/metabolism , Survival Analysis , Vinblastine/pharmacology , Young AdultABSTRACT
The prognosis for patients with glioblastoma (GB) remains grim. Concurrent temozolomide (TMZ) radiation-the cornerstone of glioma control-extends the overall median survival of GB patients by only a few months over radiotherapy alone. While these survival gains could be partly attributed to radiosensitization, this benefit is greatly minimized in tumors expressing O6-methylguanine DNA methyltransferase (MGMT), which specifically reverses O6-methylguanine lesions. Theoretically, non-O6-methylguanine lesions (i.e., the N-methylpurine adducts), which represent up to 90% of TMZ-generated DNA adducts, could also contribute to radiosensitization. Unfortunately, at concentrations attainable in clinical practice, the alkylation capacity of TMZ cannot overwhelm the repair of N-methylpurine adducts to efficiently exploit these lesions. The current therapeutic application of TMZ therefore faces two main obstacles: (i) the stochastic presence of MGMT and (ii) a blunted radiosensitization potential at physiologic concentrations. To circumvent these limitations, we are developing a novel molecule called NEO212-a derivatization of TMZ generated by coupling TMZ to perillyl alcohol. Based on gas chromatography/mass spectrometry and high-performance liquid chromatography analyses, we determined that NEO212 had greater tumor cell uptake than TMZ. In mouse models, NEO212 was more efficient than TMZ at crossing the blood-brain barrier, preferentially accumulating in tumoral over normal brain tissue. Moreover, in vitro analyses with GB cell lines, including TMZ-resistant isogenic variants, revealed more potent cytotoxic and radiosensitizing activities for NEO212 at physiologic concentrations. Mechanistically, these advantages of NEO212 over TMZ could be attributed to its enhanced tumor uptake presumably leading to more extensive DNA alkylation at equivalent dosages which, ultimately, allows for N-methylpurine lesions to be better exploited for radiosensitization. This effect cannot be achieved with TMZ at clinically relevant concentrations and is independent of MGMT. Our findings establish NEO212 as a superior radiosensitizer and a potentially better alternative to TMZ for newly diagnosed GB patients, irrespective of their MGMT status.
Subject(s)
Dacarbazine/analogs & derivatives , Drug Resistance, Neoplasm , Glioma/drug therapy , Radiation-Sensitizing Agents/therapeutic use , Temozolomide/therapeutic use , Animals , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Brain/metabolism , Brain Neoplasms/drug therapy , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Survival/drug effects , DNA Damage/drug effects , Dacarbazine/analysis , Dacarbazine/metabolism , Dacarbazine/pharmacology , Dacarbazine/therapeutic use , Drug Resistance, Neoplasm/genetics , Gas Chromatography-Mass Spectrometry , Glioma/pathology , Humans , Mice , Mice, Inbred C57BL , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Radiation-Sensitizing Agents/analysis , Radiation-Sensitizing Agents/metabolism , Radiation-Sensitizing Agents/pharmacology , Temozolomide/analysis , Temozolomide/metabolism , Temozolomide/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Temozolomide (TMZ) is the first-line treatment for Glioblastoma Multiforme (GBM). After administration, TMZ is rapidly converted into its active metabolite (MTIC). However, its pharmacological activity is reduced due MTIC low bioavailability in the brain. Since drugs' permeability through biological barriers and tumor cell membranes affects its bioavailability, the ability of MTIC to interact with the biological membranes presents a major contribution on its pharmacological properties and activity. Biomembrane models mimic the physiological conditions, allowing to predict the drug's behavior at biological membranes and its effects on drug biodistribution profiles. In this work, lipid bilayer models using liposomes were applied for the drug-membrane interaction studies. The zwitterionic phospholipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and cholesterol were chosen for the composition of the model, since they represent the major components of the membranes of GBM cells and brain capillary endothelial cell. Thus, the molecular interactions between MTIC and these models were studied by the evaluation of the partition of the drug into the phospholipid's membrane, its location within the bilayer and its effect on the fluidity of the membrane. The attained results suggest that the composition of membranes affects drugs partition, showing that drug biodistribution depends not only on its physicochemical features, but also depends on the characteristics of the membrane such as the packing of the lipid molecules. Also, MTIC exhibited low affinity to biological membranes, explaining its low bioavailability on the target cells.
Subject(s)
Antineoplastic Agents, Alkylating/metabolism , Cholesterol/metabolism , Dimyristoylphosphatidylcholine/metabolism , Glioblastoma/metabolism , Membranes, Artificial , Temozolomide/metabolism , Antineoplastic Agents, Alkylating/administration & dosage , Dacarbazine/administration & dosage , Dacarbazine/analogs & derivatives , Dacarbazine/metabolism , Drug Interactions/physiology , Glioblastoma/drug therapy , Humans , Temozolomide/administration & dosageABSTRACT
Melanoma is a highly aggressive skin cancer. A paclitaxel formulation of solid lipid nanoparticles modified with Tyr-3-octreotide (PSM) is employed to treat melanoma that highly expresses somatostatin receptors (SSTRs). PSM exerts more apoptotic and anti-invasive effects in B16F10 mice melanoma cells as compared to dacarbazine (DTIC), an approved chemotherapeutic drug for treating aggressive melanoma. Besides, PSM induces one of the biomarkers of immunogenic cell death in vitro and in vivo as confirmed by calreticulin exposure on the B16F10 cell surface. We observed a significant number of CD8 positive T cells in the tumor bed of the PSM treated group. As a result, PSM effectively reduces tumor volume in vivo as compared to DTIC. PSM also induces a favorable systemic immune response as determined in the spleen and sera of the treated animals. Importantly, PSM can reduce the number of nodule formations in the experimental lung metastasis model. Our experimentations indicate that the metronomic PSM exhibits remarkable anti-melanoma activities without any observable toxicity. This immune modulation behavior of PSM can be exploited for the therapy of melanoma and probably for other malignancies.
Subject(s)
Antineoplastic Agents, Alkylating/chemistry , Nanoparticles/chemistry , Paclitaxel/chemistry , Peptides/chemistry , Animals , Antineoplastic Agents, Alkylating/metabolism , Antineoplastic Agents, Alkylating/pharmacology , Antineoplastic Agents, Alkylating/therapeutic use , Apoptosis/drug effects , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Calreticulin/chemistry , Calreticulin/pharmacology , Cell Line, Tumor , Cytokines/metabolism , Dacarbazine/chemistry , Dacarbazine/metabolism , Dacarbazine/pharmacology , Dacarbazine/therapeutic use , Disease Models, Animal , Female , Lung Neoplasms/drug therapy , Lung Neoplasms/mortality , Lung Neoplasms/secondary , Melanoma, Experimental/drug therapy , Melanoma, Experimental/mortality , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Paclitaxel/metabolism , Paclitaxel/pharmacology , Paclitaxel/therapeutic use , Survival Rate , Tissue DistributionABSTRACT
Glioblastoma multiforme is the most lethal type of brain tumor and the established therapy only extends patients survival to approximately one year. Its first-line treatment is based on of chemotherapy with the alkylating agent temozolomide (TMZ). As many other chemotherapeutic drugs, TMZ presents several limitations as high toxicity and low bioavailability. The delivery of TMZ using poly(lactic-co-glycolic acid) nanoparticles is proposed in this work. Stable nanoparticles functionalized with a OX26 type monoclonal antibody for transferrin receptor were developed, targeting the glioblastoma tumor cells, since these cells are known for overexpressing this receptor. The release profile of TMZ from the nanoparticles was studied mimicking physiological conditions, and targeted cellular internalization was also investigated. Two glioblastoma cell lines - U215 and U87 - were used to evaluate the in vitro cytotoxicity of the drug, showing that the prepared nanocarriers enhance the anticancer activity of TMZ. The functionalization with the monoclonal antibody for transferrin receptor proved to be advantageous in enhancing the cellular internalization in glioblastoma cells.
Subject(s)
Antibodies, Monoclonal/metabolism , Antineoplastic Agents, Alkylating/pharmacology , Brain Neoplasms/drug therapy , Dacarbazine/analogs & derivatives , Drug Carriers , Glioblastoma/drug therapy , Lactic Acid/chemistry , Nanoparticles , Polyglycolic Acid/chemistry , Receptors, Transferrin/metabolism , Antibodies, Monoclonal/chemistry , Antineoplastic Agents, Alkylating/chemistry , Antineoplastic Agents, Alkylating/metabolism , Brain Neoplasms/immunology , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Dacarbazine/chemistry , Dacarbazine/metabolism , Dacarbazine/pharmacology , Dose-Response Relationship, Drug , Drug Compounding , Drug Liberation , Glioblastoma/immunology , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Kinetics , Nanotechnology , Polylactic Acid-Polyglycolic Acid Copolymer , Receptors, Transferrin/immunology , Technology, Pharmaceutical/methods , TemozolomideABSTRACT
Recent experimental data showed that the PI3K pathway contributes to resistance to temozolomide (TMZ) in paediatric glioblastoma and that this effect is reversed by combination treatment of TMZ with a PI3K inhibitor. Our aim is to assess whether this combination results in metabolic changes that are detectable by nuclear magnetic resonance (NMR) spectroscopy, potentially providing metabolic biomarkers for PI3K inhibition and TMZ combination treatment. Using two genetically distinct paediatric glioblastoma cell lines, SF188 and KNS42, in vitro 1H-NMR analysis following treatment with the dual pan-Class I PI3K/mTOR inhibitor PI-103 resulted in a decrease in lactate and phosphocholine (PC) levels (P<0.02) relative to control. In contrast, treatment with TMZ caused an increase in glycerolphosphocholine (GPC) levels (P≤0.05). Combination of PI-103 with TMZ showed metabolic effects of both agents including a decrease in the levels of lactate and PC (P<0.02) while an increase in GPC (P<0.05). We also report a decrease in the protein expression levels of HK2, LDHA and CHKA providing likely mechanisms for the depletion of lactate and PC, respectively. Our results show that our in vitro NMR-detected changes in lactate and choline metabolites may have potential as non-invasive biomarkers for monitoring response to combination of PI3K/mTOR inhibitors with TMZ during clinical trials in children with glioblastoma, subject to further in vivo validation.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Biomarkers, Tumor/metabolism , Brain Neoplasms/drug therapy , Dacarbazine/analogs & derivatives , Furans/administration & dosage , Glioblastoma/drug therapy , Pyridines/administration & dosage , Pyrimidines/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Brain Neoplasms/diagnostic imaging , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Child , Dacarbazine/administration & dosage , Dacarbazine/metabolism , Dacarbazine/pharmacology , Furans/therapeutic use , Humans , Lactic Acid/metabolism , Phosphorylcholine/metabolism , Proton Magnetic Resonance Spectroscopy/methods , Pyridines/therapeutic use , Pyrimidines/therapeutic use , Temozolomide , Treatment OutcomeABSTRACT
Gliomas are the most common and primary tumors of the central nervous system in adults. Temozolomide (TMZ) is the main drug used to treat glioma; however, prognosis remains poor for most patients. Glioma stem cells (GSCs) are thought to enable glioma initiation and evasion from immune surveillance; their immunogenicity can be determined by expression of major histocompatibility complex (MHC)-I. The present study investigated the effect of TMZ on MHC-I expression in GSCs. Glioma spheres were cultured in serum-free medium containing epidermal growth factor, basic fibroblast growth factor, and B27; MHC-I expression was detected by immunocytochemistry, quantitative real-time PCR, and flow cytometry. Nuclear factor (NF)-κB expression in glioma stem cells was detected by Western blot. TMZ enhanced MHC-I expression in GSCs, and NF-κB was activated. TMZ treatment increased MHC-I expression via modulation of NF-κB signaling in GSCs. In addition to being a chemotherapeutic agent, TMZ may also serve as an immunomodulatory agent in the treatment of glioma patients.
Subject(s)
Dacarbazine/analogs & derivatives , Genes, MHC Class I/drug effects , Glioma/drug therapy , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Dacarbazine/metabolism , Dacarbazine/pharmacology , Drug Synergism , Glioma/metabolism , Humans , NF-kappa B/drug effects , NF-kappa B/metabolism , Neoplastic Stem Cells/metabolism , Primary Cell Culture/methods , Signal Transduction/drug effects , Stem Cells/drug effects , TemozolomideABSTRACT
The blood-brain barrier (BBB) limits entry of most chemotherapeutic agents into the CNS, resulting in inadequate exposure within CNS tumor tissue. Intranasal administration is a proposed means of delivery that can bypass the BBB, potentially resulting in more effective chemotherapeutic exposure at the tumor site. The objective of this study was to evaluate the feasibility and pharmacokinetics (plasma and CSF) of intranasal delivery using select chemotherapeutic agents in a non-human primate (NHP) model. Three chemotherapeutic agents with known differences in CNS penetration were selected for intranasal administration in a NHP model to determine proof of principle of CNS delivery, assess tolerability and feasibility, and to evaluate whether certain drug characteristics were associated with increased CNS exposure. Intravenous (IV) temozolomide (TMZ), oral (PO) valproic acid, and PO perifosine were administered to adult male rhesus macaques. The animals received a single dose of each agent systemically and intranasally in separate experiments, with each animal acting as his own control. The dose of the agents administered systemically was the human equivalent of a clinically appropriate dose, while the intranasal dose was the maximum achievable dose based on the volume limitation of 1 mL. Multiple serial paired plasma and CSF samples were collected and quantified using a validated uHPLC/tandem mass spectrometry assay after each drug administration. Pharmacokinetic parameters were estimated using non-compartmental analysis. CSF penetration was calculated from the ratio of areas under the concentration-time curves for CSF and plasma (AUCCSF:plasma). Intranasal administration was feasible and tolerable for all agents with no significant toxicities observed. For TMZ, the degrees of CSF drug penetration after intranasal and IV administration were 36 (32-57) and 22 (20-41)%, respectively. Although maximum TMZ drug concentration in the CSF (Cmax) was lower after intranasal delivery compared to IV administration due to the lower dose administered, clinically significant exposure was achieved in the CSF after intranasal administration with the lower doses. This was associated with lower systemic exposure, suggesting increased efficiency and potentially lower toxicities of TMZ after intranasal delivery. For valproic acid and perifosine, CSF penetration after intranasal delivery was similar to systemic administration. Although this study demonstrates feasibility and safety of intranasal drug administration, further agent-specific studies are necessary to optimize agent selection and dosing to achieve clinically-relevant CSF exposures.
Subject(s)
Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Administration, Intranasal , Animals , Antineoplastic Agents/metabolism , Blood-Brain Barrier , Dacarbazine/administration & dosage , Dacarbazine/analogs & derivatives , Dacarbazine/metabolism , Dacarbazine/pharmacokinetics , Disease Models, Animal , Macaca mulatta , Male , Nasal Absorption , Phosphorylcholine/administration & dosage , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/metabolism , Phosphorylcholine/pharmacokinetics , Temozolomide , Valproic Acid/administration & dosage , Valproic Acid/metabolism , Valproic Acid/pharmacokineticsABSTRACT
The aim of the present work was to investigate the efficacy of temozolomide nanostructured lipid carriers (TMZ-NLCs) to enhance brain targeting via nasal route administration. The formulation was optimized by applying a four-factor, three-level Box-Behnken design. The developed formulations and the functional relationships between their independent and dependent variables were observed. The independent variables used in the formulation were gelucire (X1), liquid lipid/total lipid (X2), Tween 80 (X3), and sonication time (X4), and their effects were observed with regard to size (Y1), % drug release (Y2), and drug loading (Y3). The optimized TMZ-NLC was further evaluated for its surface morphology as well as ex vivo permeation and in vivo studies. All TMZ-NLC formulations showed sizes in the nanometer range, with high drug loading and prolonged drug release. The optimized formulation (TMZ-NLCopt) showed an entrapment efficiency of 81.64 ± 3.71%, zeta potential of 15.21 ± 3.11 mV, and polydispersity index of less than 0.2. The enhancement ratio was found to be 2.32-fold that of the control formulation (TMZ-disp). In vivo studies in mice showed that the brain/blood ratio of TMZ-NLCopt was found to be significantly higher compared to that of TMZ-disp (intranasal, intravenous). Scintigraphy images of mouse brain showed the presence of a high concentration of TMZ. The AUC ratio of TMZ-NLCopt to TMZ-disp in the brain was the highest among the organs. The findings of this study substantiate the existence of a direct nose-to-brain delivery route for NLCs.
Subject(s)
Brain/metabolism , Dacarbazine/analogs & derivatives , Nanoparticles/chemistry , Nasal Mucosa/metabolism , Animals , Dacarbazine/chemistry , Dacarbazine/metabolism , Dacarbazine/pharmacokinetics , Drug Compounding/methods , Female , Male , Mice , Polysorbates/chemistry , Radionuclide Imaging , Rats , Rats, Wistar , Solubility , TemozolomideABSTRACT
Glioblastoma multiforme (GBL) is the most common and aggressive brain neoplasm. A standard therapeutic approach for GBL involves combination therapy consisting of surgery, radiotherapy, and chemotherapy. The latter is based on temozolomide (TMZ). However, even by applying such a radical treatment strategy, the mean patient survival time is only 14.6 months. Here we review the molecular mechanisms underlying the resistance of GBL cells to TMZ including genetic and epigenetic mechanisms. Present data regarding a role for genes and proteins MGMT, IDH1/2, YB-1, MELK, MVP/LRP, MDR1 (ABCB1), and genes encoding other ABC transporters as well as Akt3 kinase in developing resistance of GBL to TMZ are discussed. Some epigenetic regulators of resistance to TMZ such as microRNA and EZH2 are reviewed.
Subject(s)
Antineoplastic Agents, Alkylating/therapeutic use , Brain Neoplasms/drug therapy , Dacarbazine/analogs & derivatives , Drug Resistance, Neoplasm , Glioblastoma/drug therapy , Animals , Antineoplastic Agents, Alkylating/chemistry , Antineoplastic Agents, Alkylating/metabolism , Blood-Brain Barrier , Brain/enzymology , Brain/metabolism , Brain/pathology , Brain Neoplasms/enzymology , Brain Neoplasms/genetics , Brain Neoplasms/therapy , Dacarbazine/metabolism , Dacarbazine/therapeutic use , Epigenesis, Genetic , Glioblastoma/enzymology , Glioblastoma/genetics , Glioblastoma/therapy , Humans , TemozolomideABSTRACT
BACKGROUND: Co-delivery of gene and anticancer drug into the same cancer cells or tissues by multifunctional nanocarriers may provide a new paradigm in cancer treatment. In this study, nanostructured lipid carriers (NLCs) were constructed as multifunctional nanomedicine for co-delivery of enhanced green fluorescence protein plasmid (DNA) and temozolomide (TMZ). METHODS: TMZ- and DNA-loaded NLCs (TMZ/DNA-NLCs) were prepared. Their particle size, zeta potential, gene-loading capacity (GL) and drug encapsulation efficiency (EE) were evaluated. In vitro cytotoxicity study TMZ/DNA-NLCs was tested in U87 malignant glioma cells (U87 MG cells). In vivo gene transfection and anti-tumor efficacy of the carriers were evaluated on mice bearing malignant glioma model. RESULTS: The optimum TMZ/DNA-NLCs formulations with the particle size of 179 nm and with a +23 mV surface charge; got 91% of GL and 83% of EE. The growth of U87 MG cells in vitro was obviously inhibited. TMZ/DNA-NLCs also displayed the highest gene transfection efficiency and the best antitumor activity than other formulations in vivo. CONCLUSION: The results demonstrated that TMZ/DNA-NLCs were efficient in selective delivery to malignant glioma cells. Also TMZ/DNA-NLCs transfer both drug and gene to the gliomatosis cerebri, enhance the antitumor capacity and gene transfection efficacy. Thus, TMZ/DNA-NLCs could prove to be a superior co-delivery nanomedicine to achieve therapeutic efficacy and this report could be a new promising strategy for treatment in malignant gliomatosis cerebri.
Subject(s)
Antineoplastic Agents/chemistry , DNA/chemistry , DNA/pharmacology , Dacarbazine/analogs & derivatives , Lipids/chemistry , Neoplasms, Neuroepithelial/drug therapy , Animals , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Chemistry, Pharmaceutical , DNA/genetics , Dacarbazine/chemistry , Dacarbazine/metabolism , Dacarbazine/pharmacology , Drug Carriers , Drug Delivery Systems , Humans , Mice , Nanomedicine , Nanostructures , Neoplasms, Neuroepithelial/chemistry , Temozolomide , TransfectionABSTRACT
Temozolomide (TMZ) is an anticancer drug used for the treatment of adult brain tumour and skin cancer. The biomolecular interaction between TMZ and DNA was investigated for the first time in this study using disposable pencil graphite electrodes (PGEs) in combination with electrochemical techniques. The surface confined interactions between TMZ and different type of nucleic acids were performed. Before/after surface confined interaction process, the oxidation signals of TMZ, guanine and adenine were measured using differential pulse voltammetry (DPV) and PGE and accordingly, the changes at the oxidation signals were evaluated. The detection limit (DL) was also estimated based on the oxidation signal of TMZ. The interaction of TMZ with single stranded poly [A], poly [G], or double stranded poly [A]-poly[T] and poly [G]-poly[C] was also explored. Moreover, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were utilized for detection the interaction between TMZ and DNA. The features of this single-use electrochemical sensor was discussed in comparison to other reports that were developed for TMZ detection.
Subject(s)
Antineoplastic Agents/metabolism , DNA/metabolism , Dacarbazine/analogs & derivatives , Disposable Equipment , Electrochemistry/instrumentation , Graphite/chemistry , Antineoplastic Agents/chemistry , Cost-Benefit Analysis , DNA/chemistry , Dacarbazine/chemistry , Dacarbazine/metabolism , Electrochemistry/economics , Electrodes , Limit of Detection , TemozolomideABSTRACT
Chemotherapeutic resistance can occur by P-glycoprotein (P-gp), a 12-transmembrane ATP-dependent drug efflux pump. Glioblastoma (GBM) has poor survival rate and uniformly acquired chemoresistance to its frontline agent, Temozolomide (TMZ). Despite much effort, overcoming TMZ resistance remains a challenge. We reported on autonomous induction of TMZ resistance by increased transcription MDR1, the gene for P-gp. This study investigated how P-gp and TMZ interact to gain resistance. Using an experimental model of Adriamycin-resistant DC3F cells (DC3F/Adx), we showed that increased P-gp caused TMZ resistance. Increasing concentrations of TMZ competed with Calcein for P-gp, resulting in reduced efflux in the DC3F/Adx cells. Three different inhibitors of P-gp reversed the resistance to TMZ in two different GBM cell lines, by increasing active Caspase 3. Molecular modeling predicted the binding sites to be the intracellular region of P-gp and also identified specific amino acids and kinetics of energy for the efflux of TMZ. Taken together, we confirmed P-gp targeting of TMZ, a crucial regulator of TMZ resistance in GBM. This study provides insights on the effectiveness by which TMZ competes with other P-gp substrates, thereby opening the door for combined targeted therapies.
Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Brain Neoplasms/metabolism , Dacarbazine/analogs & derivatives , Drug Resistance, Neoplasm , Glioblastoma/metabolism , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Animals , Antineoplastic Agents, Alkylating/metabolism , Binding, Competitive , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Caspase 3/metabolism , Cell Line, Tumor , Cricetinae , Dacarbazine/metabolism , Dacarbazine/pharmacology , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/drug effects , Fluoresceins/metabolism , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Temozolomide , Transcription, Genetic , Transcriptional Activation , Transfection , Up-RegulationABSTRACT
Here we evaluate the photosensitizer meso-tetraphenyl chlorin disulphonate (TPCS2a) in survival studies of rat glioma cancer cells in combination with the novel photochemical internalization (PCI) technique. The tested anticancer drugs were bleomycin (BLM) and temozolomide (TMZ). Glioma cells were incubated with TPCS2a (0.2 µg ml(-1), 18 h, 37 °C) before BLM or TMZ stimulation (4 h) prior to red light illumination (652 nm, 50 mW cm(-2)). The cell survival after BLM (0.5 µm)-PCI (40 s light) quantified using the MTT assay was reduced to about 25% after 24 h relative to controls, and to 31% after TMZ-PCI. The supplementing quantification by clonogenic assays, using BLM (0.1 µm), indicated a long-term cytotoxic effect: the surviving fraction of clonogenic cells was reduced to 5% after light exposure (80 s) with PCI, compared to 70% in the case of PDT. In parallel, structural and morphological changes within the cells upon light treatment were examined using fluorescence microscopy techniques. The present study demonstrates that PCI of BLM is an effective method for killing F98 glioma cells, but smaller effects were observed using TMZ following the "light after" strategy. The results are the basis for further in vivo studies on our rat glioma cancer model using PDT and PCI.
Subject(s)
Antineoplastic Agents/metabolism , Bleomycin/metabolism , Dacarbazine/analogs & derivatives , Glioma/metabolism , Photochemical Processes , Animals , Antineoplastic Agents/chemistry , Bleomycin/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/radiation effects , Dacarbazine/chemistry , Dacarbazine/metabolism , Light , Molecular Structure , Photosensitizing Agents/pharmacology , Porphyrins/pharmacology , Rats , Temozolomide , Tumor Stem Cell AssayABSTRACT
In vitro, treosulfan (TREO) has shown high effectiveness against malignant gliomas. However, a first clinical trial for newly diagnosed glioblastoma did not show any positive effect. Even though dosing and timing might have been the reasons for this failure, it might also be that TREO does not reach the brain in sufficient amount. Surprisingly, there are no published data on TREO uptake into the brain of patients, despite extensive research on this compound. An in-vitro blood-brain barrier (BBB) model consisting of primary porcine brain capillary endothelial cells was used to determine the transport of TREO across the cell monolayer. Temozolomide (TMZ), the most widely used cytotoxic drug for malignant gliomas, served as a reference. An HPLC-ESI-MS/MS procedure was developed to detect TREO and TMZ in cell culture medium. Parallel to the experimental approach, the permeability of TREO and the reference substance across the in-vitro BBB was estimated on the basis of their physicochemical properties. The detection limit was 30 nmol/l for TREO and 10 nmol/l for TMZ. Drug transport was measured in two directions: influx, apical-to-basolateral (A-to-B), and efflux, basolateral-to-apical (B-to-A). For TREO, the A-to-B permeability was lower (1.6%) than the B-to-A permeability (3.0%). This was in contrast to TMZ, which had higher A-to-B (13.1%) than B-to-A (7.2%) permeability values. The in-vitro BBB model applied simulated the human BBB properly for TMZ. It is, therefore, reasonable to assume that the values for TREO are also meaningful. Considering the lack of noninvasive, significant alternative methods to study transport across the BBB, the porcine brain capillary endothelial cell model was efficient to collect first data for TREO that explain the disappointing clinical results for this drug against cerebral tumors.
Subject(s)
Antineoplastic Agents, Alkylating/metabolism , Blood-Brain Barrier/metabolism , Busulfan/analogs & derivatives , Dacarbazine/analogs & derivatives , Endothelial Cells/metabolism , Animals , Biological Transport , Busulfan/metabolism , Capillaries/metabolism , Cells, Cultured , Dacarbazine/metabolism , Gray Matter/blood supply , Swine , TemozolomideABSTRACT
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is associated with a poor prognosis because of a lack of effective treatment options. The objective of this study was to examine a new strategy for HCC treatment, namely the use of poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor (ABT-888) together with Temozolomide (TMZ) incorporated onto magnetic nanoparticles. METHODS: Magnetic Fe3 O4 /Fe cores were encapsulated within a silica shell to facilitate the simultaneous incorporation of ABT-888 and TMZ. In vitro tests were performed with HepG2, Hep3B and PLC-PRF-5 liver tumoural cell lines and with WRL-68 liver non-tumoural cells. RESULTS: The magnetic nanocarriers were loaded simultaneously with ABT-888 and TMZ. High stability and extended release were achieved in culture medium. Confocal microscopy images showed that drug-loaded particles were uptaken and accumulated into the cytoplasm of liver tumoural cells, inducing the following effects: G2/M cell cycle arrest (P < 0.05), accumulation of DNA damage (P < 0.05), mitochondrial depolarization (P < 0.01), reduction in BCL-xL, FOS, JUND gene expression (P < 0.05), PARP-1 fragmentation, Caspase-3 activation and apoptotic cell death (P < 0.05). Interestingly, drugs loaded onto nanoparticles exhibited better efficiency than free drugs (cell death triggered by drug delivery nanosystem: 53.5% vs. 34.5% by free drugs, P = 0.01). CONCLUSIONS: These magnetic nanocompounds are able to incorporate both drugs simultaneously, enter the tumour cells and release them. ABT-888/TMZ/NPs decrease the transcription of key genes involved in tumour survival and induce apoptotic cell death in a more effective manner than is achieved by free drugs.
Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Benzimidazoles/pharmacology , Carcinoma, Hepatocellular/drug therapy , Dacarbazine/analogs & derivatives , Drug Carriers , Liver Neoplasms/drug therapy , Magnetite Nanoparticles , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerases/metabolism , Antineoplastic Agents, Alkylating/chemistry , Antineoplastic Agents, Alkylating/metabolism , Apoptosis/drug effects , Benzimidazoles/chemistry , Benzimidazoles/metabolism , Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/pathology , Chemistry, Pharmaceutical , DNA Damage , Dacarbazine/chemistry , Dacarbazine/metabolism , Dacarbazine/pharmacology , Dose-Response Relationship, Drug , Drug Combinations , Drug Synergism , G2 Phase Cell Cycle Checkpoints/drug effects , Hep G2 Cells , Humans , Liver Neoplasms/enzymology , Liver Neoplasms/pathology , Membrane Potential, Mitochondrial/drug effects , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerase Inhibitors/chemistry , Poly(ADP-ribose) Polymerase Inhibitors/metabolism , Signal Transduction/drug effects , Technology, Pharmaceutical/methods , TemozolomideABSTRACT
Focused ultrasound (FUS) exposure with the presence of microbubbles has been shown to transiently open the blood-brain barrier (BBB), and thus has potential to enhance the delivery of various kinds of therapeutic agents into brain tumors. The purpose of this study was to assess the preclinical therapeutic efficacy of FUS-BBB opening for enhanced temozolomide (TMZ) delivery in glioma treatment. FUS exposure with microbubbles was delivered to open the BBB of nude mice that were either normal or implanted with U87 human glioma cells. Different TMZ dose regimens were tested, ranging from 2.5 to 25 mg/kg. Plasma and brain samples were obtained at different time-points ranging from 0.5 to 4 hours, and the TMZ concentration within samples was quantitated via a developed LC-MS/MS procedure. Tumor progression was followed with T2-MRI, and animal survival and brain tissue histology were conducted. Results demonstrated that FUS-BBB opening caused the local TMZ accumulation in the brain to increase from 6.98 to 19 ng/mg. TMZ degradation time in the tumor core was found to increase from 1.02 to 1.56 hours. Improved tumor progression and animal survival were found at different TMZ doses (up to 15% and 30%, respectively). In conclusion, this study provides preclinical evidence that FUS-BBB opening increases the local concentration of TMZ to improve the control of tumor progression and animal survival, suggesting the potential for clinical application to improve current brain tumor treatment.