Cellular Mechanism Of Action Research Articles

Abstract 1943: PARG inhibitors exhibit synthetic lethality with XRCC1 deficiency and a cellular mechanism of action that is distinct from PARP inhibition

Abstract Poly(ADP-ribose) glycohydrolase (PARG) hydrolyzes poly(ADP-ribose) (PAR) chains that are polymerized by PARP enzymes, completing the PAR cycle. Small molecule inhibitors of PARG result in a dose dependent increase in cellular PAR after DNA damage. Here we demonstrate that depletion of XRCC1, a scaffolding protein with an essential role in base-excision repair (BER), sensitizes cancer cells to PARG inhibition. XRCC1 deficient cells exhibit increased nuclear PAR foci in response to PARG inhibition even in the absence of DNA damaging agents. Inhibition of PARP1 with RNAi or small molecule inhibitors rescues cell growth inhibition and reduces the amount of cellular PAR accumulation in PARG inhibitor treated cells. This indicates that the cellular growth inhibition is dependent upon cellular PAR levels, demonstrating selectivity of the small molecule inhibitors for PARG. We hypothesized that inhibition of PAR hydrolysis could result in depletion of cellular NAD as this could prevent recycling of PAR to NAD. Consistent with this hypothesis, PARG inhibition enhanced NAD depletion after treatment of cells with the DNA damaging agent methyl methanesulfonate (MMS). Live cell imaging of XRCC1 depleted cells treated with a PARG inhibitor revealed that cells have large membrane protrusions, similar to the morphology of cells that have been treated with a NAMPT inhibitor, which results in depletion of cellular NAD. Furthermore, addition of the NAD precursor, nicotinamide mononucleotide (NMN) rescued proliferation of PARG inhibited cells. Taken together, these data support a hypothesis in which PARG inhibitors are cytotoxic to sensitive cancer cells via depletion of NAD, ultimately starving the cell of ATP. Thus, PARG inhibition is a novel strategy for exploiting synthetic lethality in cancer cells. The defects that sensitize cancer cells to PARG inhibition are distinct from those that sensitize to PARP inhibitors, namely defects in homology directed repair. Approximately 15% of breast cancer samples exhibit low or no XRCC1 by IHC. A subset (approximately 35%) of the XRCC1 low patient samples also have defects in BRCA1, suggesting that the majority of XRCC1 low tumors may not be responsive to PARP inhibitors. Small molecule PARG inhibitors are currently being evaluated for efficacy in XRCC1 low xenograft models. Citation Format: Leenus Martin, Tzuling Cheng, Dominic I. James, Habiba Begum, Kate M. Smith, Allan Jordan, Ian Waddell, Kedar Vaidya, Marcus Fischer, Bing Yao, Jason Drummond, Leah Cleary, Ruben Martinez, James Sutton, Nandini Ravindran, James Joseph, Eleni Venetsanakos, Michael Dillon, Jeffrey H. Hager, Lisa D. Belmont. PARG inhibitors exhibit synthetic lethality with XRCC1 deficiency and a cellular mechanism of action that is distinct from PARP inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1943.

Abstract 1772: Immunotherapy of colorectal cancer by the T-cell targeted DART® protein MGD007: Cellular mechanisms of action

Abstract Introduction MGD007 (glycoprotein A33 x CD3), a DART protein designed to redirect T cells to target gpA33-expressing colon cancer, is presently undergoing clinical evaluation (NCT02238805). The gpA33 target was selected based on its universal expression profile across primary and metastatic CRC, including expression on putative cancer stem cell (CSC) populations. MGD007 activity in CRC cell cytolysis and its prolonged PK in non-human primates have previously been reported (Cancer Res 2014;74(19 Suppl): Abstract nr 669.1). Here we further characterize MGD007 cellular mechanisms associated with redirected T-cell killing, cytokine responses and modulation with steroids. Method Redirected killing assays were performed using luciferase labelled gpA33+ve Colo205 or RECA0201-GF colorectal cancer stem-like cells (CSLC) with freshly isolated PBMC or fractionated T-cell populations; Treg cells (CD4+, CD127lo, CD25+) were expanded for 14 days in presence of IL-2 and rapamycin and confirmed to be suppressive; steroids (budesonide and dexamethasone) were evaluated at pharmacologically relevant concentrations; multi-parameter FACS and ELISA were performed to determine cell surface marker expression and cytokine levels respectively. Results MGD007 displays potent redirected T-cell killing of gpA33+ve CRC cells, including complete lysis of CRC stem cell-like models. Importantly, MGD007 mediated cytolysis can be supported by various T-cell populations, including Treg cells. Following prolonged in vitro exposure to MGD007 and gpA33+ve tumor cells, expanded T cells acquire a memory phenotype and retain potent CTL activity when challenged with fresh gpA33+ve target cells; however, much decreased cytokine release was observed compared to that observed following initial T-cell exposure. The addition of dexamethasone or budesonide to freshly isolated effector cells and gpA33+ve target cells also reduces cytokine release levels to baseline in the presence of MGD007, with minimal impact observed on MGD007-mediated killing. Discussion MGD007 supports targeted lysis of CRC, including CSC subpopulations, and can leverage suppressive Tregs in addition to conventional T cells for cytolytic activity. Biological activity modulation is also feasible through induction of cytolytic Tmem cells with diminished cytokine release potential via sequential exposure to MGD007 or the simultaneous exposure to low-dose steroids. These data support further clinical development of MGD007 for the treatment of CRC patients. Citation Format: Paul A. Moore, Yinhua Yang, Kalpana Shah, Daorong Liu, Vatana Long, Douglas H. Smith, Zhuangyu Pan, Ralph Alderson, Liqin Liu, Syd Johnson, Ezio Bonvini. Immunotherapy of colorectal cancer by the T-cell targeted DART® protein MGD007: Cellular mechanisms of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1772.

Lionfish venom elicits pain predominantly through the activation of nonpeptidergic nociceptors.

The lionfish (Pterois volitans) is a venomous invasive species found in the Caribbean and Northwestern Atlantic. It poses a growing health problem because of the increase in frequency of painful stings, for which no treatment or antidote exists, and the long-term disability caused by the pain. Understanding the venom's algogenic properties can help identify better treatment for these envenomations. In this study, we provide the first characterization of the pain and inflammation caused by lionfish venom and examine the mechanisms through which it causes pain using a combination of in vivo and in vitro approaches including behavioral, physiological, calcium imaging, and electrophysiological testing. Intraplantar injections of the venom produce a significant increase in pain behavior, as well as a marked increase in mechanical sensitivity for up to 24 hours after injection. The algogenic substance(s) are heat-labile peptides that cause neurogenic inflammation at the site of injection and induction of Fos and microglia activation in the superficial layers of the dorsal horn. Finally, calcium imaging and electrophysiology experiments show that the venom acts predominantly on nonpeptidergic, TRPV1-negative, nociceptors, a subset of neurons implicated in sensing mechanical pain. These data provide the first characterization of the pain and inflammation caused by lionfish venom, as well as the first insight into its possible cellular mechanism of action.

Flavonoids interfere with NLRP3 inflammasome activation

NOD-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome is a component of innate immunity, and is responsible for producing mature IL-1β and -18. Several flavonoids were found to affect inflammasome pathway, but the mechanism of action is still obscure. To elucidate the effects on NLRP3 inflammasome pathway and to determine the structure-activity relationships, NLRP3 inflammasome in differentiated THP-1 cells was activated via treatment with monosodium urate (MSU) crystals. Levels of mature IL-1β, NLRP3 inflammasome components and apoptosis-associated speck-like protein containing a CARD (caspase recruitment domain) (ASC) oligomerization were investigated and the mechanisms of action were also elucidated. Among the 56 flavonoids initially tested, only flavone, 2′,4′-dihhydroxyflavone, 3′,4′-dichloroflavone, 4′,5,7-trihydroxyflavone (apigenin), 3,4′,5,7-tetrahydroxyflavone (kaempferol) and 3,3′,4′,5,7-pentahydroxyflavone (quercetin) significantly inhibited IL-1β production at 10 μM. Apigenin, kaempferol and 3′,4′-dichloroflavone inhibited ASC oligomerization without affecting the ASC level in cell lysates. Apigenin also inhibited absent in melanoma 2 (AIM2) inflammasome-related pathway, but not NLR family CARD domain-containing protein 4 (NLRC4) inflammasome activation. The action of apigenin on NLRP3 inflammasome activation is mediated partly via inhibition of phosphorylation of spleen tyrosine kinase/protein tyrosine kinase 2 (Syk/Pyk2) pathway. Furthermore, orally administered apigenin (100 mg/kg) strongly reduced the number of neutrophils and monocytes in MSU-induced peritonitis in mice. The present study, for the first time, demonstrated the structure-activity profiles of flavonoids in NLRP3 inflammasome activation and mechanisms of cellular action. Certain flavonoids including apigenin are expected to ameliorate the inflammatory symptoms in autoinflammatory diseases associated with NLRP3 inflammasome activation.

Symptom-modifying slow-acting drugs in the treatment of osteoarthritis: from molecule to clinical effect (a pharmacologist's view)

The paper systematizes data on the pharmacokinetics, molecular and cellular mechanisms of action of chondroprotectors (CPs), by using chondroitin sulfate (CS) and glucosamine (GA) as an example. It presents current ideas about the mechanisms of their absorption and penetration into the joint tissues during oral administration, the effect of CS on the cytoplasmic membrane of chondrocytes in osteoarthritis (OA). The paper also describes the plastic and regulatory functions of GA at the intracellular level after its penetration into the cells by glucose carriers. The combination of benefits from targeted and metabolic therapy determines the efficacy of combined CPs, the active principle of which is CS and GA.

Inhibitory effect of Ocotea quixos (Lam.) Kosterm. and Piper aduncum L. essential oils from Ecuador on West Nile virus infection

West Nile virus (WNV) is a mosquito-borne flavivirus responsible of neuroinvasive manifestations. Natural products are well-known for their biological activities and pharmaceutical application. In this study, the inhibitory effects of essential oils (EOs) of Ocotea quixos (Lam.) Kosterm. and Piper aduncum L. on WNV replication were investigated. WNV was incubated with EOs before adsorption on Vero cells, viral replication was carried out in the absence or presence of EO. Cells were exposed to EO before the adsorption of untreated-virus. GC-MS and GC-FID were used for chemical characterization of EOs. Cell protection from infection was observed for both EOs. P. aduncum EO was characterized by dillapiole as main compound (48.21%) and O. quixos EO by 1,8-cineole (39.15%). Further investigations, such as the study of molecular and cellular mechanisms of action and in vivo evaluation, should be performed on these essential oils to derive new potential drugs against WNV.

Antinociceptive Effects of the Antidepressant Phenelzine are Mediated by Context-Dependent Inhibition of Neuronal Responses in the Dorsal Horn

The putative strong anti-nociceptive properties of the antidepressant phenelzine (PLZ) have not been widely explored as a treatment for pain. Antinociceptive effects of PLZ were identified in the formalin model of tonic pain (Mifflin et al., 2016) and in allodynia associated with experimental autoimmune encephalomyelitis, (EAE) a mouse model of multiple sclerosis (Potter et al., 2016). Here, we further clarify the specific types of stimuli and contexts in which PLZ modulates nociceptive sensitivity. Our findings indicate that PLZ selectively inhibits ongoing inflammatory pain while sparing transient reflexive and acute nociception. We also investigated the cellular mechanisms of action of PLZ in the dorsal horn, and as expected of a monoamine-oxidase inhibitor, PLZ increased serotonin (5HT) immunoreactivity. We next used two approaches to test the hypothesis that PLZ inhibits the activation of spinal nociresponsive neurons. First, we evaluated the formalin-evoked protein expression of the immediate early gene, c-fos. PLZ reduced Fos expression in the superficial dorsal horn. Second, we evaluated the effects of PLZ on intracellular calcium responses to superfusion of glutamate (0.3–1.0 mM) in an ex vivo lumbar spinal cord slice preparation. Superfusion with PLZ (100–300 μM) reduced 1 mM glutamate-evoked calcium responses. This was blocked by pretreatment with the 5HT1A-receptor antagonist WAY-100,635, but not the alpha-2 adrenergic antagonist idazoxan. We conclude that PLZ exerts antinociceptive effects through a 5-HT/5HT1AR-dependent inhibition of neuronal responses within nociceptive circuits of the dorsal horn.

Isoform-selective HDAC1/6/8 inhibitors with an imidazo-ketopiperazine cap containing stereochemical diversity

A series of hydroxamic acids linked by different lengths to a chiral imidazo-ketopiperazine scaffold were synthesized. The compounds with linker lengths of 6 and 7 carbon atoms were the most potent in histone deacetylase (HDAC) inhibition, and were specific submicromolar inhibitors of the HDAC1, HDAC6 and HDAC8 isoforms. A docking model for the binding mode predicts binding of the hydroxamic acid to the active site zinc cation and additional interactions between the imidazo-ketopiperazine and the enzyme rim. The compounds were micromolar inhibitors of the MV4-11, THP-1 and U937 cancer cell lines. Increased levels of histone H3 and tubulin acetylation support a cellular mechanism of action through HDAC inhibition.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Embelin: a benzoquinone possesses therapeutic potential for the treatment of human cancer.

Natural products have been gaining recognition and are becoming a significant part of research in the area of drug development and discovery. Phytochemicals derived from these sources have been comprehensively studied and have displayed a wide range of activities against many fatal diseases including cancer. One such product that has gained recognition from its pharmacological properties and nontoxic nature is embelin, obtained from Embelia ribes. Amid all the vivid pharmacological activities, embelin has gained its prominence in the area of cancer research. Embelin binds to the BIR3 domain of XIAP, preventing the association of XIAP and caspase-9 resulting in the suppression of cell growth, proliferation and migration of various types of cancer cells. Furthermore, embelin modulates anti-apoptotic pathways by suppressing the activity of NF-κB, PI3-kinase/AKT, JAK/STAT pathway - among others. The present review summarizes the various reported effects of embelin on different types of cancer cells and highlights the cellular mechanisms of action.

The First Approved Gene Therapy Product for Cancer Ad-p53 (Gendicine): 12 Years in the Clinic.

Gendicine (recombinant human p53 adenovirus), developed by Shenzhen SiBiono GeneTech Co. Ltd., was approved in 2003 by the China Food and Drug Administration (CFDA) as a first-in-class gene therapy product to treat head and neck cancer, and entered the commercial market in 2004. Gendicine is a biological therapy that is delivered via minimally invasive intratumoral injection, as well as by intracavity or intravascular infusion. The wild-type (wt) p53 protein expressed by Gendicine-transduced cells is a tumor suppressor that is activated by cellular stress, and mediates cell-cycle arrest and DNA repair, or induces apoptosis, senescence, and/or autophagy, depending upon cellular stress conditions. Based on 12 years of commercial use in >30,000 patients, and >30 published clinical studies, Gendicine has exhibited an exemplary safety record, and when combined with chemotherapy and radiotherapy has demonstrated significantly higher response rates than for standard therapies alone. In addition to head and neck cancer, Gendicine has been successfully applied to treat various other cancer types and different stages of disease. Thirteen published studies that include long-term survival data showed that Gendicine combination regimens yield progression-free survival times that are significantly longer than standard therapies alone. Although the p53 gene is mutated in >50% of all human cancers, p53 mutation status did not significantly influence efficacy outcomes and long-term survival rate for Ad-p53-treated patients. To date, Shenzhen SiBiono GeneTech has manufactured 41 batches of Gendicine in compliance with CFDA QC/QA requirements, and 169,571 vials (1.0 × 1012 vector particles per vial) have been used to treat patients. No serious adverse events have been reported, except for vector-associated transient fever, which occurred in 50-60% of patients and persisted for only a few hours. The manufacturing accomplishments and clinical experience with Gendicine, as well as the understanding of its cellular mechanisms of action and implications, could provide valuable insights for the international gene therapy community and add valuable data to promote further developments and advancements in the gene therapy field.

An Overview of Cardenolides in Digitalis - More Than a Cardiotonic Compound.

The genus Digitalis L. containing species, commonly known as the "foxglove", is the main source of cardenolides, which have various pharmacological properties effective against certain pathological conditions including myocardial infarction, arterial hypertension, cardiac dysfunction, angina, and hypertrophy. Togehter with a prime effect of controlling the heart rhythm, many workers demonstrated that lanatoside C and some other cardiac glycosides are effective in several cancer treatments such as prostate and breast cancers. Due to digoxigenin derivatives of cardenolides, which are mainly used for medicinal purposes, such as digoxigenin, D. lanata as a main source is of great interest for commercial scale production of cardenolides in Europe. Phytochemical studies on cardenolides, naturally occurring plant secondary metabolites, have mainly focused on the species of the genus Digitalis L., as the members of this family have a high level and diverse content of cardenolides. During the last few decades, plant tissue culture techniques have been optimised for many plant species including Digitalis, however, the production capacity of cardenolides somehow failed to reach a commercially desired extent. In this review paper, the genus Digitalis is evaluated in terms of its main botanical and physiological features, traditional uses, molecular genetics and metabolomics, cellular mechanism of action, medicinal uses, clinical pharmacology, drug interactions, therapy in the management of cardiovascular disorders, potential utility of therapy in extracardiac conditions, and toxicity.

Structure-activity relationships and cellular mechanism of action of small molecules that enhance the delivery of oligonucleotides.

The pharmacological effects of antisense and siRNA oligonucleotides are hindered by the tendency of these molecules to become entrapped in endomembrane compartments thus failing to reach their targets in the cytosol or nucleus. We have previously used high throughput screening to identify small molecules that enhance the escape of oligonucleotides from intracellular membrane compartments and have termed such molecules OECs (oligonucleotide enhancing compounds). Here, we report on the structure–activity relationships of a family of OECs that are analogs of a hit that emerged from our original screen. These studies demonstrate key roles for the lipophilic aromatic groups, the tertiary nitrogen, and the carbamate moiety of the parent compound. We have also investigated the intracellular site of action of the OECs and have shown that activity is due to the release of oligonucleotides from intermediate endosomal compartments rather than from early endosomes or from highly acidic downstream compartments. At high concentrations of OECs toxicity occurs in a manner that is independent of caspases or of lysosomal cathepsins but instead involves increased plasma membrane permeability. Thus, in addition to describing specific characteristics of this family of OECs, the current study provides insights into basic mechanisms of oligonucleotide trafficking and their implications for oligonucleotide delivery.

Oncolytic Herpes Simplex Virus ICP47 Deletion Reverses Tumor Immune Evasion

Herpes simplex virus (HSV) is an enveloped, double-stranded DNA virus that has been used with modification as oncolytic viruses (OVs) against a number of tumor types. OVs represent a new class of therapeutic agents that promote anti-tumour responses through a dual mechanism of action that is dependent on selective tumor cell killing and the induction of systemic anti-tumour immunity. Among OVs, HSVs preferentially replicate in and lyse cancer cells, leading to in situ autovaccination, adaptive anti-virus and anti-tumor immunity. Suppression of antitumor immunity after OV therapy has been observed and the molecular and cellular mechanisms of action are recently reported. ICP47, a small protein produced by the herpes simplex virus, is considered as an important factor in the evasion of cellular immune responses in HSV-infected cells. Therefore, reviewing the research status of ICP47 is certainly helpful to improve the anti-tumor effect of oncolytic HSVs (oHSVs). Here, this review will focus on the following contents: 1) Anti-tumor mechanism of OVs; 2) Functions of early HSV genes; 3) The mechanism of immune escape of ICP47; 4) Recombinant HSV against cancer; 5) The functional verification of ICP47 deletion. This review highlights the current understanding of recombinant HSVs against cancers.

Antiproliferative Effects of a Triterpene-Enriched Extract from Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), on Human Lung Cancer Cells.

Ganoderma lucidum, a mushroom that has been used to treat disease in East Asia for centuries, has been shown to be effective against many types of tumors, but the exact cellular mechanism of action is unknown. In this study we examined proliferation of a lung cancer cell line after treatment with 12 concentrations of powdered G. lucidum for 24, 48, and 120 hours. Based on half-maximal inhibitory concentrations values, proliferation of the H1793 cell line seemed to be sensitive to the extract in a time- and dose-dependent manner. We used immunoblot analysis to examine the amounts of cell cycle proteins (cyclin D, Cdk4, and Cdc2) and apoptotic proteins (Bcl-xL and Bax) after treatment with a range of G. lucidum concentrations. Changes in amounts of proteins that regulate the cell cycle were consistent with longer G1 and G2 phases. Proapoptotic protein (Bax) levels increased 6.5-fold, with a commensurate increase in the Bax-to-Bcl ratio, especially at 48 and 120 hours. These results suggest that the decrease in cellular proliferation correlated with a change in both cell cycle progression and apoptosis, and that the triterpenoid in G. lucidum is the bioactive component. Further biochemical characterization of this ancient herbal remedy could hold promise for treating lung cancer.

Axonal GABAA receptors depolarize presynaptic terminals and facilitate transmitter release in cerebellar Purkinje cells.

GABAA receptors have been described in the axonal compartment of neurons; contrary to dendritic GABAA receptors, axonal GABAA receptors usually induce depolarizing responses. In this study we describe the presence of functional axonal GABAA receptors in cerebellar Purkinje cells by using a combination of direct patch-clamp recordings from the axon terminals and laser GABA photolysis. In Purkinje cells, axonal GABAA receptors are depolarizing and induce an increase in neurotransmitter release that results in a change of short-term synaptic plasticity. These results contribute to our understanding of the cellular mechanisms of action of axonal GABAA receptors and highlight the importance of the presynaptic compartment in neuronal computation. In neurons of the adult brain, somatodendritic GABAA receptors (GABAA Rs) mediate fast synaptic inhibition and play a crucial role in synaptic integration. GABAA Rs are not only present in the somatodendritic compartment, but also in the axonal compartment where they modulate action potential (AP) propagation and transmitter release. Although presynaptic GABAA Rs have been reported in various brain regions, their mechanisms of action and physiological roles remain obscure, particularly at GABAergic boutons. Here, using a combination of direct whole-bouton or perforated patch-clamp recordings and local GABA photolysis in single axonal varicosities of cerebellar Purkinje cells, we investigate the subcellular localization and functional role of axonal GABAA Rs both in primary cultures and acute slices. Our results indicate that presynaptic terminals of PCs carry GABAA Rs that behave as auto-receptors; their activation leads to a depolarization of the terminal membrane after an AP due to the relatively high cytoplasmic Cl- concentration in the axon, but they do not modulate the AP itself. Paired recordings from different terminals of the same axon show that the GABAA R-mediated local depolarizations propagate substantially to neighbouring varicosities. Finally, the depolarization mediated by presynaptic GABAA R activation augmented Ca2+ influx and transmitter release, resulting in a marked effect on short-term plasticity. Altogether, our results reveal a mechanism by which presynaptic GABAA Rs influence neuronal computation.

Cellular mechanisms of action and resistance of Plasmodium falciparum to artemisinin.

The recent reports of high failure rates and decline in in vitro sensitivity of Plasmodium falciparum to artemisinin-based combination therapies (ACTs) suggest the possibility of clinical artemisinin resistance along the Thai-Cambodian and Thai-Myanmar borders. The study investigated cellular mechanisms of action and resistance of P. falciparum to artesunate (stage specific activity, interaction with hemozoin, and anti-oxidant levels) in the two paired P. falciparum isolates (MSF046 and MSF060) collected before treatment with a 3-day artesunate-mefloquine and at the time of recrudescence. In addition, the link of these cellular mechanisms to the polymorphisms of the candidate artemisinin-resistant genes (pfatp6, pfcrt, pfmdr1, pfmrp1, and K13 propeller) was also investigated. Morphological change was observed in both pairs of the primary and recrudesced P. falciparum isolates during 12-48h of exposure to artesunate (at IC90). A marked decrease in parasite viability was found in the recrudesced isolates of both MSF046 and MSD060. The extent of the reduction (% change of baseline) in total glutathione concentrations was significantly lower in recrudesced (32.1 and 1.7%) compared with primary (45.5 and 53.7%) isolates of both MSF046 and MSF060. The extent of reduction of hemozoin content in MSF046 was significantly higher in the recrudesced (76.8%) isolate compared with the primary isolate (99.5%). For MSF060 on the other hand, increase in hemozoin content was found in the recrudesced isolate and the extent of such increase was significantly higher in recrudesced (93.1%) than the primary isolate (87.5%). Polymorphism of K13 (N458Y) together with pfmdr1 copy number correlated well with sensitivity of both isolates to artesunate. Results of this preliminary study suggests possible role of glutathione-dependent detoxification system as well as heme degradation as cellular mechanisms of action and resistance of artemisinins.

Impact of Depleting Therapeutic Monoclonal Antibodies on the Host Adaptive Immunity: A Bonus or a Malus?

Clinical responses to anti-tumor monoclonal antibody (mAb) treatment have been regarded for many years only as a consequence of the ability of mAbs to destroy tumor cells by innate immune effector mechanisms. More recently, it has also been shown that anti-tumor antibodies can induce a long-lasting anti-tumor adaptive immunity, likely responsible for durable clinical responses, a phenomenon that has been termed the vaccinal effect of antibodies. However, some of these anti-tumor antibodies are directed against molecules expressed both by tumor cells and normal immune cells, in particular lymphocytes, and, hence, can also strongly affect the host adaptive immunity. In addition to a delayed recovery of target cells, lymphocyte depleting-mAb treatments can have dramatic consequences on the adaptive immune cell network, its rebound, and its functional capacities. Thus, in this review, we will not only discuss the mAb-induced vaccinal effect that has emerged from experimental preclinical studies and clinical trials but also the multifaceted impact of lymphocytes-depleting therapeutic antibodies on the host adaptive immunity. We will also discuss some of the molecular and cellular mechanisms of action whereby therapeutic mAbs induce a long-term protective anti-tumor effect and the relationship between the mAb-induced vaccinal effect and the immune response against self-antigens.

Preferential Mitochondrial Localization of a Goniothalamin Fluorescent Derivative.

A fluorescent 2,1,3-benzothiadiazole-containing goniothalamin derivative, BTD−GTN (1), has been synthesized and successfully tested in bioimaging experiments in live cells. The fluorescent compound proved to be capable of transposing the cell membranes, indicating its subcellular localization. The use of the benzothiadiazole core as the fluorophore revealed the favored localization of the GTN analogue 1 in the cytoplasm of live cells, preferentially in the mitochondria, in line with previous results that indicated the loss of mitochondrial transmembrane potential upon treatment with GTN. The results described herein highlight the potential of the BTD–GTN hybrid structures for future studies regarding the cellular mechanism of action of this family of compounds.

Abstract DDT02-01: Discovery of GDC-0077, a highly isoform selective inhibitor of PI3Kα that promotes selective loss of mutant-p110α

Abstract PIK3CA, the gene that encodes for the p110α catalytic subunit of phosphatidylinositol-3-kinase (PI3Kα), is frequently mutated in cancer, with activating and transforming mutations of PIK3CA occurring in approximately 17% of cancers diagnosed annually. The narrow therapeutic index of PI3K inhibitors evaluated in the clinic thus far may be due to insufficient selectivity for PI3K isoform or mutant oncogenic driver. We report the results of our efforts directed at improving efficacy and safety of a class of benzoxazepin inhibitors of PI3K. A structure-, property-, and phenotype-guided strategy directed at high PI3K-isoform and PIKK-family selectivity, as well as a distinct cellular mechanism of action, culminated in the discovery of GDC-0077. Differences in primary and tertiary structure among PI3K isoforms were leveraged to achieve >300-fold selective inhibition of the PI3Kα isoform over the β, δ, and γ isoforms in biochemical assays. Very high selectivity over PIKK-family proteins such as mTOR, DNA-PK and VPS34 was also achieved through this process. In vitro and in vivo data indicate that GDC-0077 operates by a mechanism that promotes the selective degradation of “hotspot” p110α mutants (H1047R, E545K). Treatment of nude mice bearing PIK3CA-mutant breast tumor xenografts with oral GDC-0077 resulted in tumor regressions at well-tolerated doses. Together, the high degree of isoform selectivity, selective degradation of mutant-p110α, and in vivo PK/efficacy support the clinical evaluation of GDC-0077 as a treatment for patients with locally advanced or metastatic, PIK3CA-mutant cancer. Citation Format: Steven T. Staben. Discovery of GDC-0077, a highly isoform selective inhibitor of PI3Kα that promotes selective loss of mutant-p110α [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr DDT02-01. doi:10.1158/1538-7445.AM2017-DDT02-01

Urolithins impair cell proliferation, arrest the cell cycle and induce apoptosis in UMUC3 bladder cancer cells.

Ellagitannins have been gaining attention as potential anticancer molecules. However, the low bioavailability of ellagitannins and their extensive metabolization in the gastrointestinal tract into ellagic acid and urolithins suggest that the health benefits of consuming ellagitannins rely on the direct effects of their metabolites. Recently, chemopreventive and chemotherapeutic activities were ascribed to urolithins. Nonetheless, there is still a need to screen and evaluate the selectivity of these molecules and to elucidate their cellular mechanisms of action. Therefore, this work focused on the antiproliferative effects of urolithins A, B and C and ellagic acid on different human tumor cell lines. The evaluation of cell viability and the determination of the half-maximal inhibitory concentrations indicated that the sensitivity to the studied urolithins varied markedly between the different cell lines, with the bladder cancer cells (UMUC3) being the most susceptible. In UMUC3 cells, urolithin A was the most active molecule, promoting cell cycle arrest at the G2/M checkpoint, increasing apoptotic cell death and inhibiting PI3K/Akt and MAPK signaling. Overall, the present study emphasizes the chemopreventive/chemotherapeutic potential of urolithins, highlighting the stronger effects of urolithin A and its potential to target transitional bladder cancer cells.