Yanrong Qian

A Small-Molecule Inhibitor of Glucose Transporter 1 Downregulates Glycolysis, Induces Cell-Cycle Arrest, and Inhibits Cancer Cell Growth In Vitro and In Vivo

The functional and therapeutic importance of the Warburg effect is increasingly recognized, and glycolysis has become a target of anticancer strategies. We recently reported the identification of a group of novel small compounds that inhibit basal glucose transport and reduce cancer cell growth by a glucose deprivation–like mechanism. We hypothesized that the compounds target Glut1 and are efficacious in vivo as anticancer agents. Here, we report that a novel representative compound WZB117 not only inhibited cell growth in cancer cell lines but also inhibited cancer growth in a nude mouse model. Daily intraperitoneal injection of WZB117 at 10 mg/kg resulted in a more than 70% reduction in the size of human lung cancer of A549 cell origin. Mechanism studies showed that WZB117 inhibited glucose transport in human red blood cells (RBC), which express Glut1 as their sole glucose transporter. Cancer cell treatment with WZB117 led to decreases in levels of Glut1 protein, intracellular ATP, and glycolytic enzymes. All these changes were followed by increase in ATP-sensing enzyme AMP-activated protein kinase (AMPK) and declines in cyclin E2 as well as phosphorylated retinoblastoma, resulting in cell-cycle arrest, senescence, and necrosis. Addition of extracellular ATP rescued compound-treated cancer cells, suggesting that the reduction of intracellular ATP plays an important role in the anticancer mechanism of the molecule. Senescence induction and the essential role of ATP were reported for the first time in Glut1 inhibitor–treated cancer cells. Thus, WZB117 is a prototype for further development of anticancer therapeutics targeting Glut1-mediated glucose transport and glucose metabolism. Mol Cancer Ther; 11(8); 1672–82. ©2012 AACR.

The Warburg effect: evolving interpretations of an established concept.

Metabolic reprogramming and altered bioenergetics have emerged as hallmarks of cancer and an area of active basic and translational cancer research. Drastically upregulated glucose transport and metabolism in most cancers regardless of the oxygen supply, a phenomenon called the Warburg effect, is a major focuses of the research. Warburg speculated that cancer cells, due to defective mitochondrial oxidative phosphorylation (OXPHOS), switch to glycolysis for ATP synthesis, even in the presence of oxygen. Studies in the recent decade indicated that while glycolysis is indeed drastically upregulated in almost all cancer cells, mitochondrial respiration continues to operate normally at rates proportional to oxygen supply. There is no OXPHOS-to-glycolysis switch but rather upregulation of glycolysis. Furthermore, upregulated glycolysis appears to be for synthesis of biomass and reducing equivalents in addition to ATP production. The new finding that a significant amount of glycolytic intermediates is diverted to the pentose phosphate pathway (PPP) for production of NADPH has profound implications in how cancer cells use the Warburg effect to cope with reactive oxygen species (ROS) generation and oxidative stress, opening the door for anticancer interventions taking advantage of this. Recent findings in the Warburg effect and its relationship with ROS and oxidative stress controls will be reviewed. Cancer treatment strategies based on these new findings will be presented and discussed.

Oxime-based inhibitors of glucose transporter 1 displaying antiproliferative effects in cancer cells.

An analysis of the main pharmacophoric features present in the still limited number of inhibitors of glucose transporter GLUT1 led to the identification of new oxime-based inhibitors, which proved to be able to efficiently hinder glucose uptake and cell growth in H1299 lung cancer cells. The most important interactions of a representative inhibitor were indicated by a novel computational model of GLUT1, which was purposely developed to explain these results and to provide useful indications for the design and the development of new and more efficient GLUT1 inhibitors.

Biological and biomedical functions of Penta-O-galloyl-d-glucose and its derivatives

Penta-O-galloyl-d-glucose (PGG) is a simple hydrolysable tannin in plants. PGG exists in two anomeric forms, α-PGG and β-PGG. While β-PGG can be found in a wide variety of plants, α-PGG is rather rare in nature. Numerous studies with β-PGG revealed a wide variety of biological activities, such as anti-microbial and anti-cancer functions. Until recently, studies with α-PGG were limited by the lack of its availability. Since the development of an efficient chemical synthesis of the compound, several investigations have revealed its anti-diabetic, anti-cancer, and anti-platelet-coagulation functions. Based on structure–activity-relationship (SAR) studies with α-PGG, a variety of α-PGG-related novel compounds were synthesized and some of them have been shown to possess promising therapeutic activities. In this review, the authors will survey and evaluate the biological functions of PGG with a focus on α-PGG and its derivatives.

Constituents of an Extract of Cryptocarya rubra Housed in a Repository with Cytotoxic and Glucose Transport Inhibitory Effects

A new alkylated chalcone (1), a new 1,16-hexadecanediol diester (2), and eight known compounds were isolated from a dichloromethane-soluble repository extract of the leaves and twigs of Cryptocarya rubra collected in Hawaii. The structures of the new compounds were determined by interpretation of their spectroscopic data, and the absolute configurations of the two known cryptocaryanone-type flavonoid dimers, (+)-bicaryanone A (3) and (+)-chalcocaryanone C (4), were ascertained by analysis of their electronic circular dichroism and NOESY NMR spectra. All compounds isolated were evaluated against HT-29 human colon cancer cells, and, of these, (+)-cryptocaryone (5) was found to be potently cytotoxic toward this cancer cell line, with an IC50 value of 0.32 μM. This compound also exhibited glucose transport inhibitory activity when tested in a glucose uptake assay.

Salicylketoximes that target glucose transporter 1 restrict energy supply to lung cancer cells

The glucose transporter GLUT1 is frequently overexpressed in most tumor tissues because rapidly proliferating cancer cells rely primarily on glycolysis, a low‐efficiency metabolic pathway that necessitates a very high rate of glucose consumption. Because blocking GLUT1 is a promising anticancer strategy, we developed a novel class of GLUT1 inhibitors based on the 4‐aryl‐substituted salicylketoxime scaffold. Some of these compounds are efficient inhibitors of glucose uptake in lung cancer cells and have a notable antiproliferative effect. In contrast to their 5‐aryl‐substituted regioisomers, the newly synthesized compounds reported herein do not display significant binding to the estrogen receptors. The inhibition of glucose uptake in cancer cells by these compounds was further observed by fluorescence microscopy imaging using a fluorescent analogue of glucose. Therefore, blocking the ability of tumor cells to take up glucose by means of these small molecules, or by further optimized derivatives, may be a successful approach in the development of novel anticancer drugs.

Glucose- and Triglyceride-lowering Dietary Penta-O-galloyl-α-D-Glucose Reduces Expression of PPARγ and C/EBPα, Induces p21-Mediated G1 Phase Cell Cycle Arrest, and Inhibits Adipogenesis in 3T3-L1 Preadipocytes.

Plant polyphenols, such as hydrolysable tannins, are present in the human diet and known to exhibit anti-diabetic and anti-obesity activity. We previously reported that the representative hydrolysable tannin compound α-penta-galloyl-glucose (α-PGG) is a small molecule insulin mimetic that, like insulin, binds to insulin receptor (IR) and activates the IR-Akt-GLUT4 signaling pathway to trigger glucose transport and reduce blood glucose levels in db/db and ob/ob diabetic mice. However, its effects on adipogenesis and lipid metabolism were not known. In this study, high fat diet (HFD)-induced diabetic and obese mice were treated with α-PGG to determine its effects on blood glucose and triglycerides. 3T3-L1 preadipocytes were used as a cell model for identifying the anti-adipogenic activity of α-PGG at molecular and cellular levels as a first step in elucidating the mechanism of action of the compound. In vivo, oral administration of α-PGG significantly reduced levels of blood glucose, triglyceride, and insulin in HFD-induced diabetic/obese mice (P<0.05). In vitro, α-PGG inhibited the differentiation of 3T3-L1 preadipocytes into mature adipocytes. α-PGG suppressed the expression of positive adipogenic factors PPARγ C/EBPα and mTOR and augmented the negative adipogenic factor Pref-1. Furthermore, α-PGG induced upregulation of p21 and G1 phase cell cycle arrest. In contrast, adipogenic signaling pathways mediated by insulin, the cAMP response element binding protein (CREB) and glucocorticoid receptor (GR), were not inhibited. RNAi knockdown of p21 led to a 4-fold increase in triglyceride level in 3T3-L1 preadipocytes treated with MDI and α-PGG compared to regular preadipocytes. These results indicate, for the first time, that α-PGG is blood triglyceride- and glucose-lowering in HFD-induced obese and diabetic mice. It selectively inhibited some but not all major adipogenic pathways as well as the mTOR-p21-mediated cell cycle regulatory pathway. It is very likely that these apparently diverse but coordinated activities together inhibited adipogenesis. These results expand our knowledge on how PGG works in adipocytes and further confirm that α-PGG functions as an orally-deliverable natural insulin mimetic with adipogenetic modulatory functions.

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

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.

Extracellular ATP a New Player in Cancer Metabolism: NSCLC Cells Internalize ATP in vitro and in vivo Using Multiple Endocytic Mechanisms.

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. Implications: Extracellular ATP is taken up by human lung cancer cells and tumors via macropinocytosis and other endocytic processes to supplement their extra energy needs for cancer growth, survival, and drug resistance, thus providing novel targets for future cancer therapy. Mol Cancer Res; 14(11); 1087–96. ©2016 AACR.

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

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.