Diverse temporal and spatial mechanisms work, partially through Stanniocalcin-1, V-ATPase and senescence, to activate the extracellular ATP-mediated drug resistance in human cancer cells.

Introduction: Resistance to drug therapies is associated with a large majority of cancer-related deaths. ATP-binding cassette (ABC) transporter-mediated drug efflux, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs), glutathione (GSH), senescence, and vacuole-type ATPase (V-ATPase) all contribute to the resistance. We recently showed that extracellular ATP (eATP) induces and regulates EMT, CSC formation, and ABC transporters in human cancer cells and tumors. eATP also consistently upregulates Stanniocalcin-1 (STC1), a gene that significantly contributes to EMT, CSC formation, and tumor growth. We also found that eATP enhances drug resistance in cancer cells through eATP internalization mediated by macropinocytosis, leading to an elevation of intracellular ATP (iATP) levels, induction of EMT, and CSC formation. However, these factors have never been systematically investigated in the context of eATP-induced drug resistance. Methods: In this study, we hypothesized that eATP increases drug resistance via inducing ABC efflux, EMT, CSCs, STC1, and their accompanied processes such as GSH reducing activity, senescence, and V-ATPase. RNA sequencing, metabolomics, gene knockdown and knockout, and functional assays were performed to investigate these pathways and processes. Results and discussion: Our study results showed that, in multiple human cancer lines, eATP induced genes involved in drug resistance, elevated ABC transporters' efflux activity of anticancer drugs; generated transcriptomic and metabolic profiles representing a drug resistant state; upregulated activities of GSH, senescence, and V-ATPase to promote drug resistance. Collectively, these newly found players shed light on the mechanisms of eATP-induced as well as STC1- and V-ATPase-mediated drug resistance and offer potential novel targets for combating drug resistance in cancers.

First-in-Class Dual Targeting Compounds for the Management of Seizures in Glucose Transporter Type 1 Deficiency Syndrome.

The genetic disorder glucose transporter type 1 deficiency syndrome (GLUT1-DS) heavily affects the main intake of energy in tissues and determines the most relevant outcomes at the central nervous system (CNS) district, which is highly dependent on glucose. Herein, we report the design and development of a set of compounds bearing the glucosyl and galactosyl moieties. We assessed their ability to enhance the GLUT1 mediated glucose intake in non-small-cell lung cancer (NSCLC) cells and to inhibit the carbonic anhydrase (CA; EC 4.2.1.1) isoforms implicated in the physiopathology of uncontrolled seizures associated to epilepsy (i.e., I, II, IV, VA, VB, and XII). The binding mode of 8 in adduct with hCA II was determined by X-ray crystallography. Among the selected derivatives, compound 4b proved effective in suppressing the occurrence of uncontrolled seizures on the in vivo induced maximal electroshock (MES) model and thus gives sustainment of an unprecedently reported pharmacological approach for the management of GLUT1-DS associated diseases.

An emerging master inducer and regulator for epithelial-mesenchymal transition and tumor metastasis: extracellular and intracellular ATP and its molecular functions and therapeutic potential.

Despite the rapid development of therapeutic strategies in cancer treatment, metastasis remains the major cause of cancer-related death and scientific challenge. Epithelial-Mesenchymal Transition (EMT) plays a crucial role in cancer invasion and progression, a process by which tumor cells lose cell-cell adhesion and acquire increased invasiveness and metastatic activity. Recent work has uncovered some crucial roles of extracellular adenosine 5'- triphosphate (eATP), a major component of the tumor microenvironment (TME), in promoting tumor growth and metastasis. Intratumoral extracellular ATP (eATP), at levels of 100-700 µM, is 103-104 times higher than in normal tissues. In the current literature, eATP's function in promoting metastasis has been relatively poorly understood as compared with intracellular ATP (iATP). Recent evidence has shown that cancer cells internalize eATP via macropinocytosis in vitro and in vivo, promoting cell growth and survival, drug resistance, and metastasis. Furthermore, ATP acts as a messenger molecule that activates P2 purinergic receptors expressed on both tumor and host cells, stimulating downstream signaling pathways to enhance the invasive and metastatic properties of tumor cells. Here, we review recent progress in understanding eATP's role in each step of the metastatic cascade, including initiating invasion, inducing EMT, overcoming anoikis, facilitating intravasation, circulation, and extravasation, and eventually establishing metastatic colonization. Collectively, these studies reveal eATP's important functions in many steps of metastasis and identify new opportunities for developing more effective therapeutic strategies to target ATP-associated processes in cancer.

Cancer Stem Cell Formation Induced and Regulated by Extracellular ATP and Stanniocalcin-1 in Human Lung Cancer Cells and Tumors.

Cancer stem cells (CSCs) are closely associated with metastasis and epithelial mesenchymal transition (EMT). We previously reported that extracellular ATP (eATP) induces and regulates EMT in cancer cells. We recently found that the gene stanniocalcin 1 (STC1) is significantly upregulated by eATP in human non-small lung cancer (NSCLC) A549 cells; however, the relationships among eATP, CSCs, and STC1 were largely unknown. In this study, we performed gene knockdown and knockout, and a wide variety of functional assays to determine if and how eATP and STC1 induce CSCs in NSCLC A549 and H1299 cells. Our data show that, in both cultured cells and tumors, eATP increased the number of CSCs in the cancer cell population and upregulated CSC-related genes and protein markers. STC1 deletion led to drastically slower cell and tumor growth, reduced intracellular ATP levels and CSC markers, and metabolically shifted STC1-deficient cells from an energetic state to a quiescent state. These findings indicate that eATP induces and regulates CSCs at transcriptional, translational, and metabolic levels, and these activities are mediated through STC1 via mitochondria-associated ATP synthesis. These novel findings offer insights into eATP-induced CSCs and identify new targets for inhibiting CSCs.

From Transcriptomics, Metabolomics to Functional Studies: Extracellular ATP Induces TGF-ß-Like Epithelial Mesenchymal Transition in Lung Cancer Cells.

We and others previously showed that extracellular ATP (eATP) is implicated in epithelial mesenchymal transition (EMT). However, the mechanisms by which eATP induces EMT and ATP's relationship to TGF-ß, a well-known EMT inducer, are largely unclear. Also, eATP-induced EMT has never been studied at transcriptomic and metabolomics levels. Based on our previous studies, we hypothesized that eATP acts as a specific inducer and regulator of EMT at all levels in cancer cells. RNAseq and metabolomics analyses were performed on human non-small cell lung cancer (NSCLC) A549 cells treated with either eATP or TGF-ß. Bio-functional assays, such as invasion, intracellular ATP, cell proliferation, cytoskeleton remodeling, and others were conducted in NSCLC A549 and H1299 cells to validate changes observed from RNAseq and metabolomics studies. In the RNAseq study, eATP significantly enriched expressions of genes involved in EMT similarly to TGF-ß after 2 and 6 hours of treatment. Samples treated with eATP for 2 hours share 131 upregulated EMT genes with those of TGF-ß treated samples, and 42 genes at 6 hours treatment. Eleven genes, with known or unknown functions in EMT, are significantly upregulated by both inducers at both time points, have been identified. BLOC1S6, one of the 11 genes, was selected for further study. eATP induced numerous EMT-related changes in metabolic pathways, including cytoskeleton rearrangement, glycolysis, glutaminolysis, ROS, and individual metabolic changes similar to those induced by TGF-ß. Functional bioassays verified the findings from RNAseq and metabolomics that eATP EMT-like changes in A549 and H1299 cells similarly to TGF-ß. BLOC1S6 was found to be implicated in EMT. In these studies, eATP-induced EMT, at all levels examined, is similar but non-identical to that induced by TGF-ß, and functions in such a way that exogenous addition of TGF-ß is unnecessary for the induction. The study of BLOC1S6 further verified its potential roles in EMT and the RNAseq analysis results. All these strongly indicate that eATP is a multi-functional and multi-locational inducer and regulator of EMT, changing our thinking on how EMT is induced and regulated and pointing to new directions for inhibiting EMT in cancer.

Extracellular ATP and Macropinocytosis: Their Interactive and Mutually Supportive Roles in Cell Growth, Drug Resistance, and EMT in Cancer.

Macropinocytosis is one of the major mechanisms by which cancer cells uptake extracellular nutrients from tumor microenvironment (TME) and plays very important roles in various steps of tumorigenesis. We previously reported the unexpected finding that intratumoral and extracellular ATP (eATP), as one of the major drastically upregulated extracellular nutrients and messengers in tumors, is taken up by cancer cells through macropinocytosis in large quantities and significantly contributing to cancer cell growth, survival, and increased resistance to chemo and target drugs. Inhibition of macropinocytosis substantially reduced eATP uptake by cancer cells and slowed down tumor growth in vivo. More recently, we have found the eATP also plays a very important role in inducing epithelial-to-mesenchymal transition (EMT), and that macropinocytosis is an essential facilitator in the induction. Thus, macropinocytosis and eATP, working in coordination, appear to play some previously unrecognized but very important roles in EMT and metastasis. As a result, they are likely to be interactive and communicative with each other, regulating each other's activity for various needs of host tumor cells. They are also likely to be an integral part of the future new anticancer therapeutic strategies. Moreover, it is undoubted that we have not identified all the important activities coordinated by ATP and macropinocytosis. This review describes our findings in how eATP and macropinocytosis work together to promote cancer cell growth, resistance, and EMT. We also list scientific challenges facing eATP research and propose to target macropinocytosis and eATP to reduce drug resistance and slow down metastasis.

Cancer stem cells, epithelial-mesenchymal transition, ATP and their roles in drug resistance in cancer.

The cancer stem cell (CSC) state and epithelial-mesenchymal transition (EMT) activation are tightly interconnected. Cancer cells that acquire the EMT/CSC phenotype are equipped with adaptive metabolic changes to maintain low reactive oxygen species levels and stemness, enhanced drug transporters, anti-apoptotic machinery and DNA repair system. Factors present in the tumor microenvironment such as hypoxia and the communication with non-cancer stromal cells also promote cancer cells to enter the EMT/CSC state and display related resistance. ATP, particularly the high levels of intratumoral extracellular ATP functioning through both signaling pathways and ATP internalization, induces and regulates EMT and CSC. The three of them work together to enhance drug resistance. New findings in each of these factors will help us explore deeper into mechanisms of drug resistance and suggest new resistance-associated markers and therapeutic targets.

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 drug resistance, epithelial-mesenchymal transition (EMT), and metastasis. Intratumoral eATP is 103 to 104 times higher in concentration than in normal tissues. While eATP functions as a messenger to activate purinergic signaling for EMT induction, it is also internalized by cancer cells through upregulated macropinocytosis, a specific type of endocytosis, to perform a wide variety of biological functions. These functions include providing energy to ATP-requiring biochemical reactions, donating phosphate groups during signal transduction, and facilitating or accelerating gene expression as a transcriptional cofactor. ATP is readily available, and its study in cancer and other fields will undoubtedly increase. However, eATP study remains at an early stage, and unresolved questions remain unanswered before the important and versatile activities played by eATP and internalized intracellular ATP can be fully unraveled. These authors' laboratories' contributions to these early eATP studies include microscopic imaging of non-hydrolysable fluorescent ATP, coupled with high- and low-molecular weight fluorescent dextrans, which serve as macropinocytosis and endocytosis tracers, as well as various endocytosis inhibitors, to monitor and characterize the eATP internalization process. This imaging modality was applied to tumor cell lines and to immunodeficient mice, xenografted with human cancer tumors, to study eATP internalization in vitro and in vivo. This paper describes these in vitro and in vivo protocols, with an emphasis on modifying and finetuning assay conditions so that the macropinocytosis-/endocytosis-mediated eATP internalization assays can be successfully performed in different systems.

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.

BACKGROUND: 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. METHODS: Glucose uptake and MTT/resazurin assays were used to measure DRB18's inhibitory activities of glucose transport and cell viability/proliferation in human lung cancer and other cancer cell lines. Four HEK293 cell lines expressing GLUT1-4 individually were used to determine the IC50 values of DRB18's inhibitory activity of glucose transport. Docking studies were performed to investigate the potential direct interaction of DRB18 with GLUT1-4. Metabolomics analysis was performed to identify metabolite changes in A549 lung cancer cells treated with DRB18. DRB18 was used to treat A549 tumor-bearing nude mice. The GLUT1 gene was knocked out to determine how the KO of the gene affected tumor growth. RESULTS: DRB18 reduced glucose uptake mediated via each of GLUT1-4 with different IC50s, which match with the docking glidescores with a correlation coefficient of 0.858. Metabolomics analysis revealed that DRB18 altered energy-related metabolism in A549 cells by changing the abundance of metabolites in glucose-related pathways in vitro and in vivo. DRB18 eventually led to G1/S phase arrest and increased oxidative stress and necrotic cell death. IP injection of DRB18 in A549 tumor-bearing nude mice at 10 mg/kg body weight thrice a week led to a significant reduction in the tumor volume compared with mock-treated tumors. In contrast, the knockout of the GLUT1 gene did not reduce tumor volume. CONCLUSIONS: DRB18 is a potent pan-class I GLUT inhibitor in vitro and in vivo in cancer cells. Mechanistically, it is likely to bind the outward open conformation of GLUT1-4, reducing tumor growth through inhibiting GLUT1-4-mediated glucose transport and metabolisms. Pan-class I GLUT inhibition is a better strategy than single GLUT targeting for inhibiting tumor growth.

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

PURPOSE: 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. METHODS: Mouse islets and/or INS-1832/13 beta-cells were used to test the effects of our compounds on glucose-stimulated insulin secretion (GSIS), intracellular calcium [Ca2+]i using fura-2AM, glucose transport activity via a radioactive glucose uptake assay, intracellular ATP/ADP, and extracellular acidification (ECAR) and mitochondrial oxygen consumption rates (OCAR) using Seahorse metabolic analysis. RESULTS: Both compounds reduced GSIS in beta-cells without negatively affecting cell viability. The compounds primarily diminished glucose uptake into islets and beta-cells. Despite insulin-like effects in the peripheral tissues, these compounds do not act through the insulin receptor in islets. Further interrogation of the stimulus-secretion pathway showed that all the key metabolic factors involved in GSIS including ECAR, OCAR, ATP/ADP ratios, and [Ca2+]i of INS-1832/13 cells were diminished after the compound treatment. CONCLUSION: The compounds suppress glucose uptake of the beta-cells, which consequently slows down the rates of glycolysis and ATP synthesis, leading to decrease in [Ca2+]i and GSIS. The difference between adipocytes and beta-cells in effects on glucose uptake is of great interest. Further structural and functional modifications could produce new compounds with optimized therapeutic potentials for different target cells. The higher potency of synthetic 6Cl-TGQ in enhancing insulin signaling in adipocytes but lower potency in reducing glucose uptake in beta-cells compared to α-PGG suggests the feasibility of such an approach.

Isosteres of ester derived glucose uptake inhibitors.

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.

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

(+)-Digoxin (1) is a well-known cardiac glycoside long used to treat congestive heart failure and found more recently to show anticancer activity. Several known cardenolides (2-5) and two new analogues, (+)-8(9)-ß-anhydrodigoxigenin (6) and (+)-17-epi-20,22-dihydro-21α-hydroxydigoxin (7), were synthesized from 1 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 1 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 1 and the noncytotoxic derivative 7 bind differentially to Na+/K+-ATPase. The HO-12ß, HO-14ß, and HO-3'aα hydroxy groups of (+)-digoxin (1) 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 7 results in a rotation of its steroid core, which depotentiates the binding between this compound and Na+/K+-ATPase. Thus, 1 was found to inhibit Na+/K+-ATPase, but 7 did not. In addition, the cytotoxic 1 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.

The anti-diabetic effect of eight Lagerstroemia speciosa leaf extracts based on the contents of ellagitannins and ellagic acid derivatives.

Previously, we have reported the opposite effects of compounds isolated from Lagerstroemia speciosa leaves on a glucose transport (GLUT4) assay. Ellagitannins from L. speciosa activated GLUT4, while ellagic acid derivatives showed an inhibitory effect. As part of our continuing research on anti-diabetic nutritional supplements, we herein compared the anti-diabetic effects of several extracts (LE1-8) from leaves of L. speciosa using different manufacturing processes based on the contents of ellagitannins and ellagic acid derivatives. Their anti-diabetic effects were evaluated through glucose uptake and adipocyte differentiation in 3T3-L1 cells in vitro as well as alloxan induced diabetic mice in vivo. These extracts were given to mice by gavage at doses of 0.25, 1.0, and 4.0 g per kg body weight once a day for 21 consecutive days. Results showed that LE1 (1.0 g kg-1), LE3 (1.0 or 4.0 g kg-1), LE4 (1.0 or 4.0 g kg-1), LE5 (0.25 or 1.0 or 4.0 g kg-1) and LE7 (1.0 or 4.0 g kg-1) showed significant anti-diabetic effects in alloxan-induced diabetic mice as indicated by the decreased levels of fasting blood glucose, body weight, serum biomarkers, tissue weight and body fat, and increased final insulin levels. LE8 (1.0 g kg-1) showed a moderate anti-diabetic effect as illustrated by the reduced fasting blood glucose level while LE2 and LE6 showed slight effects in alloxan-induced diabetic mice. The potential correlation of the content of ellagitannins, ellagic acid derivatives, and corosolic acid with the anti-diabetic activity was discussed.

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

A new non-cytotoxic [(+)-17ß-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.

Plant-derived glucose transport inhibitors with potential antitumor activity.

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.

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

BACKGROUND: 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. METHODS: Floating cells, fencing, and transwell assays were used to show that ATP induces cell detachment, new colony formation, migration and invasion in human A549 and other lung cancer cells. Western blots were used to detect ATP-induced changes in EMT-related proteins; Confocal microscopy was used to demonstrate ATP-induced metastasis-related cell morphological changes. Inhibitors and siRNA knockdowns were used to determine P2X7's involvement in the ATP-induced EMT. CRISPR-Cas9 knockout of the SNX5 gene was used to identify macropinocytosis' roles in EMT and cancer cell growth both in vitro and in vivo. Student t-test and one-way ANOVA were used to determine statistical significance, P < 0.05 was considered significant. RESULTS: eATP potently induces expression of matrix metallopeptidases (MMPs), and detachment, EMT, migration, and invasion of lung cancer cells. The induction was independent of TGF-ß and semi-independent of P2X7 activation. eATP performs these functions not only extracellularly, but also intracellularly after being macropinocytically internalized to further enhance P2X7-mediated EMT, filopodia formation and other early steps of metastasis. The knockout of macropinocytosis-associated SNX5 gene significantly reduces macropinocytosis, slows down tumor growth, and changes tumor morphology in nude mice. CONCLUSIONS: Collectively, these results show that eATP's functions in these processes not only from outside of cancer cells but also inside after being macropinocytotically internalized. These findings shed light on eATP's initiator and effector roles in almost every step in early metastasis, which calls for rethinking and rebalancing energy equations of intracellular biochemical reactions and the Warburg effect, and identifies eATP and macropinocytosis as novel targets for potentially slowing down EMT and preventing metastasis.

Drug resistance and combating drug resistance in cancer.

Cancer is the second leading cause of death in the US. Current major treatments for cancer management include surgery, cytotoxic chemotherapy, targeted therapy, radiation therapy, endocrine therapy and immunotherapy. Despite the endeavors and achievements made in treating cancers during the past decades, resistance to classical chemotherapeutic agents and/or novel targeted drugs continues to be a major problem in cancer therapies. Drug resistance, either existing before treatment (intrinsic) or generated after therapy (acquired), is responsible for most relapses of cancer, one of the major causes of death of the disease. Heterogeneity among patients and tumors, and the versatility of cancer to circumvent therapies make drug resistance more challenging to deal with. Better understanding the mechanisms of drug resistance is required to provide guidance to future cancer treatment and achieve better outcomes. In this review, intrinsic and acquired resistance will be discussed. In addition, new discoveries in mechanisms of drug resistance will be reviewed. Particularly, we will highlight roles of ATP in drug resistance by discussing recent findings of exceptionally high levels of intratumoral extracellular ATP as well as intracellular ATP internalized from extracellular environment. The complexity of drug resistance development suggests that combinational and personalized therapies, which should take ATP into consideration, might provide better strategies and improved efficacy for fighting drug resistance in cancer.

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.

Cardiac Glycoside Constituents of Streblus asper with Potential Antineoplastic Activity.

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.

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.