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

Three new (1-3) and two known (4 and 5) cytotoxic cardiac glycosides were isolated and characterized from a medicinal plant, Streblus asper Lour. (Moraceae), collected in Vietnam, with six new analogues and one known derivative (5a-g) synthesized from (+)-strebloside (5). A preliminary structure-activity relationship study indicated that the C-10 formyl and C-5 and C-14 hydroxy groups and C-3 sugar unit play important roles in the mediation of the cytotoxicity of (+)-strebloside (5) against HT-29 human colon cancer cells. When evaluated in NCr nu/nu mice implanted intraperitoneally with hollow fibers facilitated with either MDA-MB-231 human breast or OVCAR3 human ovarian cancer cells, (+)-strebloside (5) showed significant cell growth inhibitory activity in both cases, in the dose range 5-30 mg/kg.

Cardiac Glycoside Constituents of Streblus asper with Potential Antineoplastic Activity.

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.

Glucose, a key nutrient utilized by human cells to provide cellular energy and a carbon source for biomass synthesis, is internalized in cells via glucose transporters that regulate glucose homeostasis throughout the human body. Glucose transporters have been used as important targets for the discovery of new drugs to treat cancer, diabetes, and heart disease, owing to their abnormal expression during these disease conditions. Thus far, several glucose transport inhibitors have been used in clinical trials, and increasing numbers of natural products have been characterized as potential anticancer agents targeting glucose transport. The present review focuses on natural product glucose transport inhibitors of plant origin, including alkaloids, flavonoids and other phenolic compounds, and isoprenoids, with their potential antitumor properties also discussed.

Plant-derived glucose transport inhibitors with potential antitumor activity.

A new non-cytotoxic [(+)-17β-hydroxystrebloside (1)] and two known cytotoxic [(+)-3'-de-O-methylkamaloside (2) and (+)-strebloside (3)] cardiac glycosides were isolated and identified from the combined flowers, leaves, and twigs of Streblus asper collected in Vietnam, with the absolute configuration of 1 established from analysis of its ECD and NMR spectroscopic data and confirmed by computational ECD calculations. A new 14,21-epoxycardanolide (3a) was synthesized from 3 that was treated with base. A preliminary structure-activity relationship study indicated that the C-14 hydroxy group and the C-17 lactone unit and the established conformation are important for the mediation of the cytotoxicity of 3. Molecular docking profiles showed that the cytotoxic 3 and its non-cytotoxic analogue 1 bind differentially to Na/K-ATPase. Compound 3 docks deeply in the Na/K-ATPase pocket with a sole pose, and its C-10 formyl and C-5, C-14, and C-4' hydroxy groups may form hydrogen bonds with the side-chains of Glu111, Glu117, Thr797, and Arg880 of Na/K-ATPase, respectively. However, 1 fits the cation binding sites with at least three different poses, which all depotentiate the binding between 1 and Na/K-ATPase. Thus, 3 was found to inhibit Na/K-ATPase, but 1 did not. In addition, the cytotoxic and Na/K-ATPase inhibitory 3 did not affect glucose uptake in human lung cancer cells, against which it showed potent activity, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na/K-ATPase but not by interacting with glucose transporters.

Cytotoxic and non-cytotoxic cardiac glycosides isolated from the combined flowers, leaves, and twigs of Streblus asper.

(+)-Digoxin () is a well-known cardiac glycoside long used to treat congestive heart failure and found more recently to show anticancer activity. Several known cardenolides (-) and two new analogues, (+)-8(9)-β-anhydrodigoxigenin () and (+)-17--20,22-dihydro-21α-hydroxydigoxin (), were synthesized from  and evaluated for their cytotoxicity toward a small panel of human cancer cell lines. A preliminary structure-activity relationship investigation conducted indicated that the C-12 and C-14 hydroxy groups and the C-17 unsaturated lactone unit are important for  to mediate its cytotoxicity toward human cancer cells, but the C-3 glycosyl residue seems to be less critical for such an effect. Molecular docking profiles showed that the cytotoxic  and the noncytotoxic derivative  bind differentially to Na/K-ATPase. The HO-12β, HO-14β, and HO-3'aα hydroxy groups of (+)-digoxin () may form hydrogen bonds with the side-chains of Asp121 and Asn122, Thr797, and Arg880 of Na/K-ATPase, respectively, but the altered lactone unit of  results in a rotation of its steroid core, which depotentiates the binding between this compound and Na/K-ATPase. Thus,  was found to inhibit Na/K-ATPase, but  did not. In addition, the cytotoxic  did not affect glucose uptake in human cancer cells, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na/K-ATPase but not by interacting with glucose transporters.

Na/K-ATPase-Targeted Cytotoxicity of (+)-Digoxin and Several Semisynthetic Derivatives.

Glucose transporters (GLUTs) facilitate glucose uptake and are overexpressed in most cancer cells. Inhibition of glucose transport has been shown to be an effective method to slow the growth of cancer cells both in vitro and in vivo. We have previously reported on the anticancer activity of an ester derived glucose uptake inhibitor. Due to the hydrolytic instability of the ester linkage we have prepared a series of isosteres of the ester moiety. Of all of the isosteres prepared, the amine linkage showed the most promise. Several additional analogues of the amine-linked compounds were also prepared to improve the overall activity.

Isosteres of ester derived glucose uptake inhibitors.

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.

Pratik Shriwas

Department of Biological Sciences,
Molecular and Cellular Biology Program,
Edison Biotechnology Institute

Ohio University

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

Pratik Shriwas

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Sciences,Molecular and Cellular Biology Program,Edison Biotechnology Institute

Ohio University

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

Department of Biological Sciences,
Molecular and Cellular Biology Program,
Edison Biotechnology Institute