Adenosine 5-triphosphate

Adenosine 5 '-triphosphate (ATP) is a multifunctional nucleotide that acts as a coenzyme in cells. ATP is responsible for transporting chemical energy for metabolic reactions in cells and is often referred to as the "monetary molecular unit" of energy transfer in cells. ATP is produced by photophosphorylation and cellular respiration. ATP is utilized by enzymes and structural proteins in many cellular reactions, including biosynthesis, motility, and cell division. A molecule of ATP contains three phosphate groups and is made from inorganic phosphate groups and adenosine diphosphate (ADP) or adenosine monophosphate (AMP) by ATP synthesis. A metabolic reaction that uses ATP as an energy source to convert it back to a precursor. As a result, ATP is constantly recycled in the body, and in the human body, about its body weight is changed over and over again every day.

Scientists believe that mitochondria evolved from bacteria captured by ancient eukaryotic host cells. Mitochondria take ADP and inorganic phosphate as reactants and regenerate ATP through oxidative phosphorylation. ATP is used as a receptor to phosphorylate proteins and lipids in signal transduction pathways catalyzed by kinases. And adenylate cyclase uses ATP to make cyclic AMP, the second signaling molecule. Cells use the ratio of ATP to AMP to determine how much energy is available and manipulate metabolic pathways that produce and consume ATP. In addition to its role in energy metabolism and communication, ATP is also incorporated into nucleic acids catalyzed by polymerase during DNA replication and transcription.

In the structure of THE ATP molecule, the purine base (adenine) is attached to the 1 'carbon atom of the five-carbon sugar (ribose), and the three phosphate groups are attached to the 5' carbon atom of the five-carbon sugar. The interaction between ATP, ADP, and AMP is the addition and removal of these phosphate groups. When ATP is involved in DNA synthesis, ribonucleotide reductase first turns ribose into deoxyribose.

ATP was discovered by Karl Lohmann in 1929, but it took years for science to determine its correct structure. In 1941, Fritz Albert Lipmann suggested that ATP was the main molecule responsible for energy transfer in cells. ATP was first synthesized artificially by Alexander Todd in 1948.

Deaggravating effects of adenosine triphosphate on malignant phenotype of human gastric cancer cells

Tumor tissue generally contains many cell subsets with phenotypic differences. M17 human gastric cancer single cell clone subline is a fast growing, poorly differentiated and malignant cell line isolated from human gastric cancer cell line (MGC-803). Gastric cancer in China has a high incidence, clinical characteristics are not obvious, not easy to early detection, lack of major markers and effective treatment drugs. Adenosine triphosphate (ATP) has a significant inhibitory effect on the proliferation of human gastric cancer cells. The phenotypic changes of M17 human gastric cancer cells treated with ATP were observed to explore the anticancer mechanism of ATP.

Materials and Methods

1. Cell culture and treatment: M17 single cell clone subline was isolated from maternal line MGC-803 by limited dilution method. After 15 generations, it still maintained rapid proliferation, poor differentiation and other malignant phenotypes. M17 cells (104/ mL) were inoculated in a culture flask containing 15% calf serum RPMI 1640, and ATP (0.23 mg/ml) was added the next day as the experimental group. Control cells were not treated with ATP. Each group was set with 2 vials of cells, the culture medium was changed daily (the experimental group contained ATP), and the cells were counted with a white blood cell counter twice a day. The above experiments were repeated twice, and the mean value was taken to calculate the inhibition rate and draw the growth curve.

2. Scanning electron microscopy sample preparation: the two groups of cells (caps) were fixed by 2% glutaraldehyde and osmium, then replaced by iso-amyl acetate, dried by critical point method, gilded with 1B-5 ion plating instrument, and observed under scanning electron microscopy.

3. Lucifer yellow (LY) fluorescent dye transfer method: The density saturated two groups of single-layer cultured cells were marked with A sharp knife (0.05%LY/PBS) for 3 minutes. The dye solution was washed away, and the order of fluorescence transmission from the labeled cells to adjacent cell layers was immediately examined under a fluorescence microscope, indicating the communication function of intercellular junctions.

4. Restaining of microfilaments with vinculin, a plaque binding protein: The cells were washed once by PBS, fixed in 2% formaldehyde /PBS for 3 min, and then extracted with 0.5%TritonX-100/PBS for 30 min. The cells reacted with Vinculin mab (Sigma Company) and FITC-sheep anti-mouse IgG. The microfilament was dyed with the fluorescent dye Rhodamine and Podopsin (Mol Probes Co., LTD.) which specifically bound F-actin, and nucleated with DAPI(4, 6-Diamidino-2-phyenylindol, Sigma Co., LTD.), and sealed with 60% glycerol PBS. The camera was observed and photographed under Olympus fluorescence microscope.

The results of

1. Inhibition of proliferation: M17 single cell clonal subline had stable malignant phenotype and rapid proliferation after 15 generations. After the action of ATP, the proliferation rate slowed down obviously. After 24 hours, the cell number of the control group was 3.6×104/ mL, and the cell number of the experimental group was 3.1×104/ mL, and the inhibition rate of proliferation was 13%. After 48 hours, the cell number of the control group was 7.16×104/ mL, and the cell number of the experimental group was 4.06×104/ml, and the inhibition rate of proliferation was 43%. After 72 hours, the cell number of the control group was 10.28×104/ mL, and the cell number of the experimental group was 4.18×104/ml, and the inhibition rate of proliferation was 67%. After 96 hours, the cell number of the control group was 14.45×104/ mL, and the cell number of the experimental group was 4.56×104/ml, and the inhibition rate of proliferation was 87%. These results indicated that ATP could significantly inhibit the proliferation of M17 cells.

2. Obvious changes of microvilli on membrane surface: M17 cell surface was densely covered with microvilli (Figure 1). After 48 hours of ATP treatment, M17 cell membrane surface was smooth and flat, and the microvilli disappeared (Figure 2), showing phenotypic characteristics of normal cells.

3. Recovery of gap junctional intercellular communication (GJIC) function: THE gap junctional intercellular communication function of M17 was defective, and LY fluorescent dye remained on the scratch wound and did not transmit to the adjacent cell layer (Figure 3). In M17 cells treated with ATP (0.23 mg/ mL) for 48 h, LY fluorescence was transmitted from the labeled scratch to adjacent cell layers for 3-4 layers (FIG. 4), indicating that ATP promoted the recovery of intercellular junction communication function in M17.

4. Recombination of microfilaments framework and expression of membrane-bonded porphyroprotein-neuxin: the microfilaments in normal human gastric mucosa cells form bundles of fibers, which are thick and dense and crisscross through the whole cytoplasmic area to form a network. The end of stress fiber terminates in the adhesion area between membrane and substrate, and the adhesion spots are teardrop shaped by Vinculin fluorescence staining. The microfilament skeleton of M17 was destroyed, stress fibers disappeared, and f-actin corpuscles were revealed by photolitide fluorescence (FIG. 5). However, in M17 cell membrane treated with ATP for 48 h, the filaments were thick and crisscrossed throughout the whole cytoplasmic region (Figure 6). M17 cells showed sparse spots by Vinculin fluorescence staining, and the adhesive spots disappeared (FIG. 7). However, the sticky spots of M17 cells treated with ATP for 48 h by Vinculin fluorescence staining were arranged in orderly order on the inner surface of the membrane (Figure 8), which was similar to the sticky spots of normal gastric mucosa cells.