ABSTRACT
Although cancer is a chronic disease, most of the in vitro experiments to assess the effectiveness of intervention are performed in hours or a few days. Moreover, none of the available methodologies to measure cell proliferation are adapted to provide information about the growth kinetic during and after treatment. Thus, the objective of this work is to provide a guide to assess long-term changes in cell population size to be used mainly in cancer research. Cumulative population doubling (CPD) graphs based on cell counting for in vitro or tumor volume for in vivo assays were used to calculate four parameters: relative end CPD (RendCPD), to quantify the end point analysis of proliferation; relative area under curve (rAUC), to describe the global chronic effect of a treatment; relative time to cross a threshold (RTCT), to indicate the delay in cell population recovery produced by a treatment; and relative proliferation rate (RPR), to describe the relative regrowth velocity of the cells that survived after treatment. These parameters describe not only the acute and chronic effects of a treatment but also the behavior of cells that are not eliminated by the treatment, providing crucial information about the growth kinetic of the surviving population. Moreover, the proposed analysis allowed the grouping of independent CPD experiments quantified at different time points and even the direct comparison of in vitro and in vivo experiments. Therefore, this new way to analyze long-term outcomes provides a global view of the effectiveness of an intervention, as an important tool for long-term studies.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Glioma/drug therapy , Glioma/pathology , Guidelines as Topic , Animals , Area Under Curve , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Models, Theoretical , Time Factors , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVE: Glioblastomas are a kind of cancer with high resistance to treatments, requiring more efficient alternatives of treatment. X-linked inhibitor of apoptosis (XIAP) is highly expressed in gliomas and, due to its inhibition of caspases, can participate in resistance to therapy. Here we test the sensitization of glioma cells with XIAP gene knockdown (KD) to drugs used in chemotherapy. METHODS: We silenced XIAP expression in U87MG glioblastoma using stable shRNA, and cells were treated with taxol, BCNU, temozolomide, cisplatin, etoposide, resveratrol (Rsv), vincristine and doxorubicin. We analyzed cell viability, cell cycle, apoptosis and senescence. RESULTS: XIAP KD cells were more sensitive to etoposide, Rsv, vincristine and doxorubicin compared to wild-type (WT) cells. Doxorubicin 1 µM and vincristine 100 nM induced higher activation of caspases after 24 h and doxorubicin induced a higher degree of senescence induction in XIAP KD cells in relation to WT cells. Phospho-p53 and phospho-H2Ax Western blot indicate subsequent DNA damage as an important effector of doxorubicin-induced death. CONCLUSIONS: This study suggests that XIAP inhibitors may sensitize gliomas to certain drugs and induce death and that the mechanisms of sensitization involve apoptosis, senescence and p53 signaling.
Subject(s)
Central Nervous System Neoplasms/drug therapy , Central Nervous System Neoplasms/genetics , Glioma/drug therapy , Glioma/genetics , X-Linked Inhibitor of Apoptosis Protein/genetics , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Caspases/metabolism , Cellular Senescence/drug effects , Cellular Senescence/genetics , DNA Damage , Doxorubicin/pharmacology , Drug Resistance, Neoplasm , Etoposide/pharmacology , Gene Knockdown Techniques , Humans , RNA, Small Interfering , Tumor Suppressor Protein p53/metabolism , Vincristine/pharmacology , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
Ecto-5'-nucleotidase (eNT/CD73, E.C.3.1.3.5) is a glycosyl phosphatidylinositol (GPI)-linked cell-surface protein with several functions, including the local generation of adenosine from AMP, with the consequent activation of adenosine receptors and the salvaging of extracellular nucleotides. It also apparently functions independently of this activity, e.g., in the mediation of cell-cell adhesion. Liver fibrosis can be considered as a dynamic and integrated cellular response to chronic liver injury and the activation of hepatic stellate cells (HSCs) plays a role in the fibrogenic process. eNT/CD73 and adenosine are reported to play an important role in hepatic fibrosis in murine models. Knockdown of eNT/CD73 leads to an increase in mRNA expression of tissue non-specific alkaline phosphatase (TNALP), another AMP-degrading enzyme and thus no alteration is seen in the total ecto-AMPase activity of the cell. eNT/CD73 knockdown also leads to changes in the expression of collagen I and a clear alteration of cell migration. We suggest that eNT/CD73 protein expression controls cell migration and collagen expression in a mechanism independent of changes in nucleotide metabolism.
Subject(s)
5'-Nucleotidase/deficiency , Cell Movement/physiology , Collagen Type I/genetics , Hepatic Stellate Cells/cytology , RNA, Messenger/metabolism , 5'-Nucleotidase/biosynthesis , 5'-Nucleotidase/genetics , 5'-Nucleotidase/metabolism , Amino Acid Sequence , Animals , Cell Line, Tumor , Cell Proliferation , Collagen Type I/metabolism , Gene Knockdown Techniques , Hepatic Stellate Cells/enzymology , Hepatic Stellate Cells/metabolism , Humans , Liver Cirrhosis/enzymology , Liver Cirrhosis/genetics , Liver Cirrhosis/pathology , Mice , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , TransfectionABSTRACT
Hypophosphatasia is caused by deficiency of activity of the tissue-nonspecific alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization. Enzyme replacement therapy (ERT) with partially purified plasma enzyme was attempted but with little clinical improvement. Attaining clinical effectiveness with ERT for hypophosphatasia may require delivering functional TNSALP enzyme to bone. We tagged the C-terminal-anchorless TNSALP enzyme with an acidic oligopeptide (a six or eight residue stretch of L-Asp), and compared the biochemical properties of the purified tagged and untagged enzymes derived from Chinese hamster ovary cell lines. The specific activities of the purified enzymes tagged with the acidic oligopeptide were the same as the untagged enzyme. In vitro affinity experiments showed the tagged enzymes had 30-fold higher affinity for hydroxyapatite than the untagged enzyme. Lectin affinity chromatography for carbohydrate structure showed little difference among the three enzymes. Biodistribution pattern from single infusion of the fluorescence-labeled enzymes into mice showed delayed clearance from the plasma up to 18 h post infusion and the amount of tagged enzyme retained in bone was 4-fold greater than that of the untagged enzyme. In vitro mineralization assays with the bone marrow from a hypophosphatasia patient using each of the three enzymes in the presence of high concentrations of pyrophosphate provided evidence of bone mineralization. These results show the anchorless enzymes tagged with an acidic oligopeptide are delivered efficiently to bone and function bioactively in bone mineralization, at least in vitro. They suggest potential advantages for use of these tagged enzymes in ERT for hypophosphatasia, which should be explored.
