fluorescence-microscopy-for-atp-internalization-mediated

Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as ...

Ohio University

Nick translation is a commonly used method for labeling DNA to make DNA hybridization probes. In this approach, the use of DNase I to generate nicks in double-stranded DNA presents an inherent drawback, because the enzyme's high rate of reaction causes significant fragmentation and shortening of the hybridization probes. Based on our recent findings regarding the nucleolytic activity of the dipeptide seryl-histidine (Ser-His) and generation of free 3' hydroxyl and 5' phosphate groups at the cleavage sites of the substrate DNA by Ser-His, it was hypothesized that this disadvantage may be overcome by using Ser-His in place of DNase I as an alternative DNA nicking agent. In this study we demonstrate that like DNase I, Ser-His randomly nicks DNA, but the dipeptide has a much lower rate of reaction that enables more complete labeling of the DNA probes with less fragmentation. DNA probes labeled through nick translation using Ser-His as the DNA nicking agent were consistently larger in size and exhibited significantly higher specific activities, and enhanced hybridization signals in Southern blot analyses compared to control DNA probes that were made using DNase I as the nicking agent. Furthermore, the degree of nicking and consequently the quality of the probes could be easily controlled by adjusting the temperature and time of the Ser-His nicking reaction. These results affirm our hypothesis that Ser-His can serve as an alternative DNA nicking agent in nick translation to yield superior DNA probes and hybridization results and suggest the possible general utility of Ser-His for wide range of biological and biomedical applications that require more moderated nicking of nucleic acids. Based upon these and computer modeling results of Ser-His, a mechanism of action is proposed to explain how Ser-His may nick DNA.

Seryl-histidine as an alternative DNA nicking agent in nick translation yields superior DNA probes and hybridizations.

Obesity is a major risk factor for Syndrome X and type II diabetes (T2D). However, most antidiabetic drugs that are hypoglycemic also promote weight gain, thus alleviating one symptom of T2D while aggravating a major risk factor that leads to T2D. Adipogenesis, the differentiation and proliferation of adipocytes, is a major mechanism leading to weight gain and obesity. It is highly desirable to develop pharmaceuticals and treatments for T2D that reduce blood glucose levels without inducing adipogenesis in patients. Previously, we reported that an extract from Lagerstroemia speciosa L. (banaba) possessed activities that both stimulated glucose transport and inhibited adipocyte differentiation in 3T3-L1 cells. Using glucose uptake assays and Western/Northern blot analyses as major tools and 3T3-L1 cells as a model, we showed that the banaba extract (BE) with tannin removed was devoid of the 2 activities, and tannic acid (TA), a major component of tannins, had the same 2 activities as BE. Inhibitors known to abolish insulin-induced glucose transport also blocked TA-induced glucose transport. We further detected that TA induced phosphorylation of the insulin receptor (IR) and Akt, as well as translocation of glucose transporter 4 (GLUT 4), the protein factors involved in the signaling pathway of insulin-mediated glucose transport. We also demonstrated that TA inhibited the expression of key genes for adipogenesis. Differences between samples with or without TA in all of the quantitative assays were significant (P < 0.05). These results suggest that TA may be useful for the prevention and treatment of T2D and its associated obesity. TA may have the potential to become the lead compound in the development of new types of antidiabetic pharmaceuticals that are able to reduce blood glucose levels without increasing adiposity.

Tannic acid stimulates glucose transport and inhibits adipocyte differentiation in 3T3-L1 cells.

Insulin mimetics from natural sources are potential therapeutics that can act alone or supplement insulin and other anti-diabetic drugs in the prevention and treatment of diabetes. We recently reported the insulin-like glucose transport stimulatory activity of tannic acid (TA) in 3T3-L1 adipocytes. In this study, we find that chemically synthesized 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose (beta-PGG), one of the components of TA, as well as its natural anomer alpha-PGG possess activity. Mechanistic studies in adipocytes with alpha-PGG, the more potent of the two anomers, reveal that inhibitors that block the insulin-mediated glucose transport, including one that inhibits the insulin receptor (IR), also completely abolish the glucose transport activated by alpha-PGG. In addition, alpha-PGG induces phosphorylation of the IR and Akt, activates PI 3-kinase, and stimulates membrane translocation of GLUT 4. Receptor binding studies indicate that alpha-PGG binds to the IR and affects the binding between insulin and IR by reducing the maximum binding of insulin to IR without significantly altering the binding affinity of insulin to IR. Western blotting analysis of the products of a cross-linking reaction suggests that alpha-PGG may bind to IR at a site located on the alpha-subunit of the receptor. Animal studies demonstrate that PGG reduces blood glucose levels and improves glucose tolerance in diabetic and obese animals. Our results suggest that PGG may serve as a model for the development of new types of anti-diabetic and anti-metabolic syndrome therapeutics.

Natural anti-diabetic compound 1,2,3,4,6-penta-O-galloyl-D-glucopyranose binds to insulin receptor and activates insulin-mediated glucose transport signaling pathway.

Type 2 diabetes (T2D) has become an epidemic worldwide while T1D remains a great medical challenge. Insulin receptor (IR) signaling activators could alleviate hyperglycemia, reduce the burden on the pancreas, and contribute to prevention and treatment of both types of diabetes. Previously, we reported the synthesis and identification of a natural antidiabetic compound α-penta-galloyl-glucose (α-PGG). Subsequent studies led to the identification of an α-P6GG derivative, 6-chloro-6-deoxy-1,2,3,4-tetra-O-galloyl-α-D-glucopyranose (6Cl-TGQ). Here, we report that 6Cl-TGQ not only induced rapid and long-lasting glucose uptake comparable to insulin in adipocytes but also reduced high blood glucose levels to near normal and significantly decreased plasma insulin levels and improved glucose tolerance performance in high-fat diet-induced T2D mice when administered orally at 5 mg/kg once every other day. Moreover, a single gavage of 6Cl-TGQ at 10 mg/kg induced rapid and sharp decline of blood glucose in streptozotocin-induced T1D mice. Our studies further indicated that 6Cl-TGQ activated IR signaling in cell models and insulin-responsive tissues of mice. 6Cl-TGQ-induced Akt phosphorylation was completely blocked by IR and PI3K inhibitors, while the induced glucose uptake was blocked by the same compounds and a Glut4 inhibitor. Receptor binding studies indicated that 6Cl-TGQ bound to IR with a higher affinity than α-PGG. Importantly, 6Cl-TGQ, unlike insulin, selectively induced phosphorylation of IR without activating IGF1R or its signaling and did not increase cancer cell proliferation. These results indicate that 6Cl-TGQ is a potent orally efficacious compound with low carcinogenic potential and may contribute to the prevention and treatment of T1D and T2D.

Orally efficacious novel small molecule 6-chloro-6-deoxy-1,2,3,4-tetra-O-galloyl-α-D-glucopyranose selectively and potently stimulates insulin receptor and alleviates diabetes.

ATP plays central roles in cancer metabolism and the Warburg effect. Intratumoral ATP concentrations are up to 10(4) times higher than those of interstitial ATP in normal tissues. However, extracellular ATP is not known to enter cancer cells. Here we report that human A549 lung cancer cells internalized extracellular ATP by macropinocytosis as demonstrated by colocalization of a nonhydrolyzable fluorescent ATP and a macropinocytosis tracer high-molecular-weight dextran, as well as by a macropinocytosis inhibitor study. Extracellular ATP also induced increase of intracellular ATP levels, without involving transcription and translation at significant levels, and cancer cells' resistance to ATP-competitor anticancer drugs, likely through the mechanism of ATP internalization. These findings, described for the first time, have profound implications in ATP-sharing among cancer cells in tumors and highlight a novel anticancer target. 

Extracellular ATP is internalized by macropinocytosis and induces intracellular ATP increase and drug resistance in cancer cells.

Intratumoral extracellular ATP concentrations are 1000 times higher than those in normal tissues of the same cell origin. However, whether or not cancer cells use the abundant extracellular ATP was unknown until we recently reported that cancer cells internalize ATP. The internalized ATP was found to substantially increase intracellular ATP concentration and promote cell proliferation and drug resistance in cancer cells. Here, using a nonhydrolyzable fluorescent ATP (NHF-ATP), radioactive and regular ATP, coupled with high and low molecular weight dextrans as endocytosis tracers and fluorescence microscopy and ATP assays, cultured human NSCLC A549 and H1299 cells as well as A549 tumor xenografts were found to internalize extracellular ATP at concentrations within the reported intratumoral extracellular ATP concentration range. In addition to macropinocytosis, both clathrin- and caveolae-mediated endocytosis significantly contribute to the ATP internalization, which led to an approximately 30% (within 45 minutes) or more than 50% (within 4 hours) increase in intracellular ATP levels after ATP incubation. This increase could not be accounted for by either purinergic receptor signaling or increased intracellular ATP synthesis rates in the ATP-treated cancer cells. These new findings significantly deepen our understanding of the Warburg effect by shedding light on how cancer cells in tumors, which are heterogeneous for oxygen and nutrition supplies, take up extracellular ATP and use the internalized ATP to perform multiple previously unrecognized functions of biological importance. They strongly suggest the existence of ATP sharing among cancer and stromal cells in tumors and simultaneously identify multiple new anticancer targets.

Extracellular ATP a New Player in Cancer Metabolism: NSCLC Cells Internalize ATP In Vitro and In Vivo Using Multiple Endocytic Mechanisms.

Cancer cells are able to uptake extracellular ATP (eATP) via macropinocytosis to elevate intracellular ATP (iATP) levels, enhancing their survival in drug treatment. However, the involved drug resistance mechanisms are unknown. Here we investigated the roles of eATP as either an energy or a phosphorylating molecule in general drug resistance mediated by ATP internalization and iATP elevation. We report that eATP increased iATP levels and promoted drug resistance to various tyrosine kinase inhibitors (TKIs) and chemo-drugs in human cancer cell lines of five cancer types. In A549 lung cancer cells, the resistance was downregulated by macropinocytosis inhibition or siRNA knockdown of PAK1, an essential macropinocytosis enzyme. The elevated iATP upregulated the efflux activity of ABC transporters in A549 and SK-Hep-1 cells as well as phosphorylation of PDGFRα and proteins in the PDGFR-mediated Akt-mTOR and Raf-MEK signaling pathways in A549 cells. Similar phosphorylation upregulations were found in A549 tumors. These results demonstrate that eATP induces different types of drug resistance by eATP internalization and iATP elevation, implicating the ATP-rich tumor microenvironment in cancer drug resistance, expanding our understanding of the roles of eATP in the Warburg effect and offering new anticancer drug resistance targets.

Extracellular ATP, as an energy and phosphorylating molecule, induces different types of drug resistances in cancer cells through ATP internalization and intracellular ATP level increase.

Extracellular ATP (eATP) was shown to induce epithelial-mesenchymal transition (EMT), a very important early process in metastasis, in cancer cells via purinergic receptor signaling. However, the exact induction mechanisms are far from fully known. We previously described that eATP is internalized by cancer cells in vitro and in vivo by macropinocytosis in human non-small cell lung cancer A549 and other cancer cells, drastically elevates intracellular ATP levels, enhances cell proliferation and resistance to anticancer drugs. In this study, we tested the hypothesis that eATP and macropinocytosis-internalized eATP also induces EMT and other early steps of metastasis.

Extracellular and macropinocytosis internalized ATP work together to induce epithelial-mesenchymal transition and other early metastatic activities in lung cancer.

Previously we showed that natural compound α-penta-galloyl-glucose (α-PGG) and its synthetic derivative 6-chloro-6-deoxy-1,2,3,4-tetra-O-galloyl-α-D-glucopyranose (6Cl-TGQ) act to improve insulin signaling in adipocytes by increasing glucose transport. In this study, we investigated the mechanism of actions of α-PGG and 6Cl-TGQ on insulin secretion.

Natural Compound α-PGG and Its Synthetic Derivative 6Cl-TGQ Alter Insulin Secretion: Evidence for Diminishing Glucose Uptake as a Mechanism.

Cancer cells drastically increase the uptake of glucose and glucose metabolism by overexpressing class I glucose transporters (GLUT1-4) to meet their energy and biomass synthesis needs and are very sensitive and vulnerable to glucose deprivation. Although targeting glucose uptake via GLUTs has been an attractive anticancer strategy, the relative anticancer efficacy of multi-GLUT targeting or single GLUT targeting is unclear. Here, we report DRB18, a synthetic small molecule, is a potent anticancer compound whose pan-class I GLUT inhibition is superior to single GLUT targeting.

A small-molecule pan-class I glucose transporter inhibitor reduces cancer cell proliferation in vitro and tumor growth in vivo by targeting glucose-based metabolism.

Article	Year
Seryl-histidine as an alternative DNA nicking agent in nick translation yields superior DNA probes and hybridizations. Bioorganic & medicinal chemistry\| PubMed ID: 11814854	2002
Tannic acid stimulates glucose transport and inhibits adipocyte differentiation in 3T3-L1 cells. The Journal of nutrition\| PubMed ID: 15671208	2005
Natural anti-diabetic compound 1,2,3,4,6-penta-O-galloyl-D-glucopyranose binds to insulin receptor and activates insulin-mediated glucose transport signaling pathway. Biochemical and biophysical research communications\| PubMed ID: 16137651	2005
Orally efficacious novel small molecule 6-chloro-6-deoxy-1,2,3,4-tetra-O-galloyl-α-D-glucopyranose selectively and potently stimulates insulin receptor and alleviates diabetes. Journal of molecular endocrinology\| PubMed ID: 23549408	2013
Extracellular ATP is internalized by macropinocytosis and induces intracellular ATP increase and drug resistance in cancer cells. Cancer letters\| PubMed ID: 24973521	2014
Extracellular ATP a New Player in Cancer Metabolism: NSCLC Cells Internalize ATP In Vitro and In Vivo Using Multiple Endocytic Mechanisms. Molecular cancer research : MCR\| PubMed ID: 27578770	2016
Extracellular ATP, as an energy and phosphorylating molecule, induces different types of drug resistances in cancer cells through ATP internalization and intracellular ATP level increase. Oncotarget\| PubMed ID: 29152126	2017
Extracellular and macropinocytosis internalized ATP work together to induce epithelial-mesenchymal transition and other early metastatic activities in lung cancer. Cancer cell international\| PubMed ID: 31582910	2019
Natural Compound α-PGG and Its Synthetic Derivative 6Cl-TGQ Alter Insulin Secretion: Evidence for Diminishing Glucose Uptake as a Mechanism. Diabetes, metabolic syndrome and obesity : targets and therapy\| PubMed ID: 33658814	2021
A small-molecule pan-class I glucose transporter inhibitor reduces cancer cell proliferation in vitro and tumor growth in vivo by targeting glucose-based metabolism. Cancer & metabolism\| PubMed ID: 33771231	2021

Yunsheng Li

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