Cysteine Restriction in Murine L929 Fibroblasts as an Alternative Strategy to Methionine Restriction in Cancer Therapy

Methionine restriction (MetR) is an efficient method of amino acid restriction (AR) in cells and organisms that induces low energy metabolism (LEM) similar to caloric restriction (CR). The implementation of MetR as a therapy for cancer or other diseases is not simple since the elimination of a single amino acid in the diet is difficult. However, the in vivo turnover rate of cysteine is usually higher than the rate of intake through food. For this reason, every cell can enzymatically synthesize cysteine from methionine, which enables the use of specific enzymatic inhibitors. In this work, we analysed the potential of cysteine restriction (CysR) in the murine cell line L929. This study determined metabolic fingerprints using mass spectrometry (LC/MS). The profiles were compared with profiles created in an earlier work under MetR. The study was supplemented by proliferation studies using D-amino acid analogues and inhibitors of intracellular cysteine synthesis. CysR showed a proliferation inhibition potential comparable to that of MetR. However, the metabolic footprints differed significantly and showed that CysR does not induce classic LEM at the metabolic level. Nevertheless, CysR offers great potential as an alternative for decisive interventions in general and tumour metabolism at the metabolic level.

Dual activity of PD-L1 targeted Doxorubicin immunoliposomes promoted an enhanced efficacy of the antitumor immune response in melanoma murine model

Background The immunomodulation of the antitumor response driven by immunocheckpoint inhibitors (ICIs) such as PD-L1 (Programmed Death Ligand-1) monoclonal antibody (α-PD-L1) have shown relevant clinical outcomes in a subset of patients. This fact has led to the search for rational combinations with other therapeutic agents such as Doxorubicin (Dox), which cytotoxicity involves an immune activation that may enhance ICI response. Therefore, this study aims to evaluate the combination of chemotherapy and ICI by developing Dox Immunoliposomes functionalized with monovalent-variable fragments (Fab’) of α-PD-L1. Results Immunoliposomes were assayed in vitro and in vivo in a B16 OVA melanoma murine cell line over-expressing PD-L1. Here, immune system activation in tumor, spleen and lymph nodes, together with the antitumor efficacy were evaluated. Results showed that immunoliposomes bound specifically to PD-L1+ cells, yielding higher cell interaction and Dox internalization, and decreasing up to 30-fold the IC50, compared to conventional liposomes. This mechanism supported a higher in vivo response. Indeed, immunoliposomes promoted full tumor regression in 20% of mice and increased in 1 month the survival rate. This formulation was the only treatment able to induce significant (p < 0.01) increase of activated tumor specific cytotoxic T lymphocytes at the tumor site. Conclusion PD-L1 targeted liposomes encapsulating Dox have proved to be a rational combination able to enhance the modulation of the immune system by blocking PD-L1 and selectively internalizing Dox, thus successfully providing a dual activity offered by both, chemo and immune therapeutic strategies. Graphic Abstract

Metabolic Fingerprinting of Murine L929 Fibroblasts as a Cell-Based Tumour Suppressor Model System for Methionine Restriction

Since Otto Warburg reported in 1924 that cancer cells address their increased energy requirement through a massive intake of glucose, the cellular energy level has offered a therapeutic anticancer strategy. Methionine restriction (MetR) is one of the most effective approaches for inducing low-energy metabolism (LEM) due to the central position in metabolism of this amino acid. However, no simple in vitro system for the rapid analysis of MetR is currently available, and this study establishes the murine cell line L929 as such a model system. L929 cells react rapidly and efficiently to MetR, and the analysis of more than 150 different metabolites belonging to different classes (amino acids, urea and tricarboxylic acid cycle (TCA) cycles, carbohydrates, etc.) by liquid chromatography/mass spectrometry (LC/MS) defines a metabolic fingerprint and enables the identification of specific metabolites representing normal or MetR conditions. The system facilitates the rapid and efficient testing of potential cancer therapeutic metabolic targets. To date, MS studies of MetR have been performed using organisms and yeast, and the current LC/MS analysis of the intra- and extracellular metabolites in the murine cell line L929 over a period of 5 days thus provides new insights into the effects of MetR at the cellular metabolic level.

„IN VITRO“ AND „IN VIVO“ ANTITUMOR ACTIVITY OF PLATINUM(II) COMPLEXES WITH MALONIC ACID ON BREAST AND COLORECTAL CARCINOMA CELL LINES

Four Pt(II) complexes of the general formula [Pt(L)(5,6-epoxy-1,10-phen)], where L is anion of malonic (mal, Pt1), 2-methylmalonic (Me-mal, Pt2), 2,2-dimethylmalonic (Me2-mal, Pt3) or 1,1- cyclobutanedicarboxylic (CBDCA, Pt4) acid while 5,6-epoxy-1,10-phen is bidentately coordinated 5,6-epoxy-5,6-dihydro-1,10-phenanthroline were synthesized and characterized by elemental microanalysis, IR, UV-Vis and NMR (1H and 13C) spectroscopic techniques. In vitro anticancer activity of novel platinum(II) complexes have been investigated on human and murine cancer cell lines, as well as normal murine cell line by MTT assay. The obtained results indicate that studied platinum(II) complexes exhibited strong cytotoxic activity against murine breast carcinoma cells (4T1), human (HCT116) and murine (CT26) colorectal carcinoma cells. Complex Pt3 display stronger selectivity toward carcinoma cells in comparison to other tested platinum(II) complexes exhibiting beneficial antitumor activity mainly via the induction of apoptosis, as well as inhibition of cell proliferation and migration. Further study showed that Pt3 complex also carry significant in vivo antitumor activity in orthotopical 4T1 tumor model without detected liver, kidney, lung, and heart toxicity. All results imply that these novel platinum(II) complexes have a good anti-tumor effect on breast and colorectal cancer in vivo and in vitro and the affinity to become possible candidates for treatment in anticancer therapy.

In vitro α-amylase and α-glucosidase Inhibition, Antioxidant, Anti- Inflammatory Activity and GC-MS Profiling of Avicennia alba Blume

Background: Avicennia alba Blume, is a well-known mangrove plant used in traditional medicinal practices for several human ailments. Objective: The study aimed at evaluation of antidiabetic, antioxidant, anti-inflammatory and cytotoxic activities of A. alba ethanolic leaf (AAL) and bark (AAB) extract along with phytochemical investigation. Methods: In vitro antidiabetic study was done by α-amylase, α-glucosidase enzyme inhibition assay; antioxidant study by DPPH, ABTS, superoxide, and metal chelating assays, antiinflammatory study by protein denaturation assay. The cytotoxicity study was done on TC1 murine cell line. Further, GC-MS analysis was carried out for AAL extracts. Results: AAL exhibited better antidiabetic activities with IC50 values of 1.18 and 0.87 mg/ml against α-amylase and α-glucosidase enzymes respectively. The AAL exhibited better ABTS, superoxide scavenging and metal chelating potential with IC50 values of 0.095, 0.127 and 0.444 mg/ml. However, AAB showed higher DPPH scavenging potential with IC50 value of 0.163 mg/ml. The AAL also exhibited higher protein denaturation potential with IC50 value of 0.370 mg/ml. The bark extract exhibited better cytotoxic activity as compared to leaf extracts on the TC1 murine cell line. The phytochemical study revealed higher total phenol (25.64 mg GAE/g), flavonoid (205.09 mg QE/g), and tannin content (251.17 mg GAE/g) in AAL. The GC-MS analysis revealed the presence of several compounds in AAL extract. Conclusion: The result of the present study highlights the antidiabetic, antioxidant and cytotoxic activities of mangrove plant Avicennia alba.

Mechanical loading of tissue engineered skeletal muscle prevents dexamethasone induced myotube atrophy

Skeletal muscle atrophy as a consequence of acute and chronic illness, immobilisation, muscular dystrophies and aging, leads to severe muscle weakness, inactivity and increased mortality. Mechanical loading is thought to be the primary driver for skeletal muscle hypertrophy, however the extent to which mechanical loading can offset muscle catabolism has not been thoroughly explored. In vitro 3D-models of skeletal muscle provide a controllable, high throughput environment and mitigating many of the ethical and methodological constraints present during in vivo experimentation. This work aimed to determine if mechanical loading would offset dexamethasone (DEX) induced skeletal muscle atrophy, in muscle engineered using the C2C12 murine cell line. Mechanical loading successfully offset myotube atrophy and functional degeneration associated with DEX regardless of whether the loading occurred before or after 24 h of DEX treatment. Furthermore, mechanical load prevented increases in MuRF-1 and MAFbx mRNA expression, critical regulators of muscle atrophy. Overall, we demonstrate the application of tissue engineered muscle to study skeletal muscle health and disease, offering great potential for future use to better understand treatment modalities for skeletal muscle atrophy.

METTL15 interacts with the assembly intermediate of murine mitochondrial small ribosomal subunit to form m4C840 12S rRNA residue

Mammalian mitochondrial ribosomes contain a set of modified nucleotides, which is distinct from that of the cytosolic ribosomes. Nucleotide m4C840 of the murine mitochondrial 12S rRNA is equivalent to the dimethylated m4Cm1402 residue of Escherichia coli 16S rRNA. Here we demonstrate that mouse METTL15 protein is responsible for the formation of m4C residue of the 12S rRNA. Inactivation of Mettl15 gene in murine cell line perturbs the composition of mitochondrial protein biosynthesis machinery. Identification of METTL15 interaction partners revealed that the likely substrate for this RNA methyltransferase is an assembly intermediate of the mitochondrial small ribosomal subunit containing an assembly factor RBFA.

Establishment of a FDC-P1 murine cell line with human KIT N822K gene overexpression

The mechanism of resistance of leukemia cells to chemotherapeutic drugs remains poorly understood. New model systems for studying the processes of malignant transformation of hematopoietic cells are needed. Based on cytokine-dependent murine acute myeloid leukemia (AML) FDC-P1 cells, we generated a new cell line with ectopic expression of the KIT gene encoding mutant human receptor tyrosine kinase (N822K). We investigated the role played by overexpression of the mutant KIT in the survival of leukemia cells and their sensitivity to therapeutic drugs. We also generated a co-culture system consisting of FDC-P1 murine leukemia cells and a HS-5 human stromal cell line. Our data can be used for a further comprehensive analysis of the role of KIT N822K mutation in the cellular response to anti-leukemic drugs, growth factors, and cytokines. These data are of interest in the development of new effective therapeutic approaches to the treatment of acute leukemia.