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Biochemical Pharmacology (v.78, #2)

Graphical Abstracts Contents (pp. e1-e4).

Editorial Advisory Board (pp. iii).

ABINs: A20 binding inhibitors of NF-κB and apoptosis signaling by Lynn Verstrepen; Isabelle Carpentier; Kelly Verhelst; Rudi Beyaert (pp. 105-114).

ABINs have been described as three different proteins (ABIN-1, ABIN-2, ABIN-3) that bind the ubiquitin-editing nuclear factor-κB (NF-κB) inhibitor protein A20 and which show limited sequence homology. Overexpression of ABINs inhibits NF-κB activation by tumor necrosis factor (TNF) and several other stimuli. Similar to A20, ABIN-1 and ABIN-3 expression is NF-κB dependent, implicating a potential role for the A20/ABIN complex in the negative feedback regulation of NF-κB activation. Adenoviral gene transfer of ABIN-1 has been shown to reduce NF-κB activation in mouse liver and lungs. However, ABIN-1 as well as ABIN-2 deficient mice exhibit only slightly increased or normal NF-κB activation, respectively, possibly reflecting redundant NF-κB inhibitory activities of multiple ABINs. Other functions of ABINs might be non-redundant. For example, ABIN-1 shares with A20 the ability to inhibit TNF-induced apoptosis and as a result ABIN-1 deficient mice die during embryogenesis due to TNF-dependent fetal liver apoptosis. On the other hand, ABIN-2 is required for optimal TPL-2 dependent extracellularly regulated kinase activation in macrophages treated with TNF or Toll-like receptor ligands. ABINs have recently been shown to contain an ubiquitin-binding domain that is essential for their NF-κB inhibitory and anti-apoptotic activities. In this context, ABINs were proposed to function as adaptors between ubiquitinated proteins and other regulatory proteins. Alternatively, ABINs might disrupt signaling complexes by competing with other ubiquitin-binding proteins for the binding to specific ubiquitinated targets. Altogether, these findings implicate an important role for ABINs in the regulation of immunity and tissue homeostasis.

Keywords: Abbreviations; aa; amino acids; ABIN; A20-binding inhibitor of NF-κB; AHD; ABIN homology domain; Ang; angiopoietin; AUF1; AU-rich element binding factor 1; EGF; epidermal growth factor; ERK; extracellularly regulated kinase; HIV; human immunodeficiency virus; IκB; inhibitor of κB; IKK; IκB kinase; IL; interleukin; LPS; lipopolysaccharide; LZ; leucine zipper; NBD; NEMO-binding domain; NF-κB; nuclear factor κB; PI3K; phosphoinositide-3 kinase; TLR; Toll-like receptor; PIC; pre-integration complex; PSGL-1; P-selectin glycoprotein ligand 1; TNF; tumor necrosis factor; TNFAIP3; tumor necrosis factor alpha induced protein 3; TPA; 12-O-tetradecanoylphorbol-13-acetate; UBD; ubiquitin-binding domainInflammation; NF-κB; Apoptosis; ABIN; A20

G-quadruplex compounds and cis-platin act synergistically to inhibit cancer cell growth in vitro and in vivo by Mekala Gunaratnam; Colin Green; João Bruno Moreira; Adam D. Moorhouse; Lloyd R. Kelland; John E. Moses; Stephen Neidle (pp. 115-122).

Senescent cells after 1 week treatment with a combination of cis-platin and the acridine compound AS1410.The ability of two structurally diverse telomeric G-quadruplex-binding compounds to synergise the action of cis-platin has been investigated in two cancer cell lines. One compound is a trisubstituted acridine compound AS1410, a close analogue of BRACO-19, and the other is a non-polycyclic compound synthesised using click chemistry and containing two triazole rings. Both compounds produce growth arrest at sub-cytotoxic concentrations in the two cell lines (MCF7 and A549), with behaviour consistent with telomere targeting mechanisms. Synergistic behaviour was observed in both cell lines with both compounds in combination with cis-platin, but only when the ratio of AS1410:cis-platin is >1. In vivo tumour xenograft studies with the A549 lung cancer model and the trisubstituted acridine compound AS1410 showed only a modest anti-tumour effect when administered alone, but produced rapid and highly significant decreases in tumour volume when administered in combination with cis-platin.

Keywords: G-quadruplex; Ligand; Telomere; Cis-platin; Synergy

A nuclear budding mechanism in transiently arrested cells generates drug-sensitive and drug-resistant cells by Sylvia Mansilla; Marc Bataller; José Portugal (pp. 123-132).

HCT116 (p53^+/+) human colon carcinoma cells treated with nanomolar concentrations of doxorubicin underwent transient senescence, synthesized DNA, showed endopolyploidization, increased their size and became multinucleated without a significant increase in mitosis. Nuclei underwent a budding process that involved the release of buds outside the nuclear membrane, and some of the buds seemed to escape from the polyploid cells. A clonogenic assay showed that some cells proliferated following the initial treatment. In general, cells ensuing after budding were not resistant to a variety of drugs, although some of them turned out to be resistant, indicating a potential selective advantage. Nuclear budding was accompanied by changes in protein levels in the giant cells, including inhibition of p53 and enhanced expression of p21^WAF1 and the meiosis-related Mos. The buds might be a mechanism for the segregation and elimination of redundant DNA, or for generating viable aneuploid cells with a potentially extended life span.

Keywords: Aneuploidy; HCT116 cells; p21; ^WAF; Mitotic catastrophe; Neosis; Senescence

Zoledronic acid determines S-phase arrest but fails to induce apoptosis in cholangiocarcinoma cells by Antonello A. Romani; Silvia Desenzani; Marina M. Morganti; Silvia La Monica; Angelo F. Borghetti; Paolo Soliani (pp. 133-141).

Zoledronic acid (ZOL) induces an S-phase arrest by altering cell cycle regulators and allowing survival of cholangiocarcinoma cells by changing the delicate balance between anti- and pro-apoptotic proteins.Cholangiocarcinoma is the second most common primary hepatic neoplasia and the only curative therapy is surgical resection or liver transplantation. Biphosphonates (BPs) are an emerging class of drugs widely used to treat bone diseases and also appear to possess direct antitumor activity. In two human cholangiocarcinoma cell lines (TFK-1 and EGI-1) we investigated, for the first time, the activity of zoledronic acid by determining proliferation, cell cycle analysis and apoptosis.The results obtained indicate that zoledronic acid induces cell-narrowing and growth inhibition, both reversed by 25μM GGOH, and significantly affects the colony-forming ability of these cells. The inhibition by zoledronic acid of Rap1A prenylation was reversed in cell co-treated with GGOH. At 10–50μM zoledronic acid exerted an S-phase cell cycle arrest which was confirmed by changes in the level of cyclins and of regulators p27^KIP1 and pRb. Interestingly, the expression level of cyclin A (putative S-phase marker) shows a dose-dependent increment in contrast to the decrement of cyclin D1 (putative G1 phase marker). However, neither hypodiploid cells nor cleaved PARP or caspase-3 was detected. The lack of TP53 or loss of its function, the large constitutive expressions of anti-apoptotic proteins Bcl-xL and HSP27 together with the low level of the pro-apoptotic Bax are the likely factors which protect cells from apoptosis.In conclusion, our study indicates that zoledronic acid induces S-phase arrest and cell-narrowing, both reversed by GGOH and, by changing the delicate balance between pro- and anti-apoptotic proteins, allows survival of cholangiocarcinoma cells.

Keywords: Cholangiocarcinoma; Zoledronic acid; Cyclins; Cell cycle regulators; Bcl-xL; GGOH

Hsp90 inhibitors, GA and 17AAG, lead to ER stress-induced apoptosis in rat histiocytoma by Aftab Taiyab; Amere S. Sreedhar; Ch. Mohan Rao (pp. 142-152).

Heat shock protein 90 (Hsp90) is a major molecular chaperone that plays an essential role in the maintenance of several signaling molecules, most of which are oncogenic kinases. Hsp90 inhibition by specific inhibitors leads to destabilization and loss of activity of such proteins, thereby leading to inhibition of multiple signaling cascades. Due to this, Hsp90 has emerged as an important target for the treatment of cancer. Inhibition of Hsp90 has been reported to induce apoptosis in certain cancer cell types. However, the molecular details of induction of apoptosis upon Hsp90 inhibition are not understood. We have investigated the effect of Hsp90 inhibition on a non-adherent rat histiocytoma cell line, BC-8, using geldanamycin and 17-Allylamino-17-demethoxygeldanamycin. We show that Hsp90 inhibition induces ER stress, which leads to disruption of mitochondrial homeostasis, leading to apoptosis. Induction of ER stress leads to increased expression of ER chaperones, Grp78 and Grp94, cleavage of caspase-12 and increase in cytoplasmic calcium. We show that calcium and Bax are responsible for the decrease in mitochondrial membrane potential (Δ ψ_m), thereby leading to the release of cytochrome c and activation of caspase-9. Moreover, calcium chelator and over-expression of Bcl-2 is able to confer protection against apoptosis upon Hsp90 inhibition. We conclude that inhibition of Hsp90 leads to ER stress-induced mitochondria-mediated apoptosis and that Bax and Ca²⁺ play an important role in mitochondrial damage.

Keywords: Hsp90; ER stress; Bax; Ca; ²⁺; Mitochondrial membrane potential

Nilotinib (AMN107, Tasigna^®) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters by Amit K. Tiwari; Kamlesh Sodani; Si-Rong Wang; Ye-Hong Kuang; Charles R. Ashby Jr.; Xiang Chen; Zhe-Sheng Chen (pp. 153-161).

Nilotinib, a BCR-Abl tyrosine kinase inhibitor (TKI), was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia positive chronic myelogenous leukemia. Recently, it was shown that several human multidrug resistance (MDR) ATP-binding cassette (ABC) proteins could be modulated by specific TKIs. MDR can produce cancer chemotherapy failure, typically due to overexpression of ABC transporters, which are involved in the extrusion of therapeutic drugs. Here, we report for the first time that nilotinib potentiates the cytotoxicity of widely used therapeutic substrates of ABCG2, such as mitoxantrone, doxorubicin, and ABCB1 substrates including colchicine, vincristine, and paclitaxel. Nilotinib also significantly enhances the accumulation of paclitaxel in cell lines overexpressing ABCB1. Similarly, nilotinib significantly increases the intracellular accumulation of mitoxantrone in cells transfected with ABCG2. Furthermore, nilotinib produces a concentration-dependent inhibition of the ABCG2-mediated transport of methotrexate (MTX), as well as E₂17βG a physiological substrate of ABCG2. Uptake studies in membrane vesicles overexpressing ABCG2 have indicated that nilotinib inhibits ABCG2 similar to other established TKIs as well as fumitremorgin C. Nilotinib is a potent competitive inhibitor of MTX transport by ABCG2 with a K_i value of 0.69±0.083μM as demonstrated by kinetic analysis of nilotinib. Overall, our results indicate that nilotinib could reverse ABCB1- and ABCG2-mediated MDR by blocking the efflux function of these transporters. These findings may be used to guide the design of present and future clinical trials with nilotinib, elucidating potential pharmacokinetic interactions. Also, these findings may be useful in clinical practice for cancer combination therapy with nilotinib.

Keywords: Abbreviations; MDR; multidrug resistance; ABC; ATP-binding cassette; ABCB1 (P-gp); P-glycoprotein; ABCG2; also called BCRP (breast cancer resistance protein)/MXR (mitoxantrone resistance protein); ABCC1 (MRP1); multidrug resistance protein 1; EGFR; epidermal growth factor receptor; HER; human epidermal receptor; TKI; tyrosine kinase inhibitor; PBS; phosphate-buffered saline; FTC; fumitremorgin C; E; ₂; 17βG; estradiol 17-β-; d; -glucuronide; MX; mitoxantrone; MTX; methotrexateNilotinib; ABC transporters; ABCB1; ABCG2; BCR-Abl; Tyrosine kinase inhibitor; Multidrug resistance

Molecular pharmacology and antitumor activity of Zalypsis^® in several human cancer cell lines by Juan F.M. Leal; Verónica García-Hernández; Victoria Moneo; Alberto Domingo; Juan Antonio Bueren-Calabuig; Ana Negri; Federico Gago; María José Guillén-Navarro; Pablo Avilés; Carmen Cuevas; Luis Francisco García-Fernández; Carlos María Galmarini (pp. 162-170).

Zalypsis^® is a new synthetic alkaloid tetrahydroisoquinoline antibiotic that has a reactive carbinolamine group. This functionality can lead to the formation of a covalent bond with the amino group of selected guanines in the DNA double helix, both in the absence and in the presence of methylated cytosines. The resulting complex is additionally stabilized by the establishment of one or more hydrogen bonds with adjacent nucleotides in the opposite strand as well as by van der Waals interactions within the minor groove. Fluorescence-based thermal denaturation experiments demonstrated that the most favorable DNA triplets for covalent adduct formation are AGG, GGC, AGC, CGG and TGG, and these preferences could be rationalized on the basis of molecular modeling results. Zalypsis^®–DNA adducts eventually give rise to double-strand breaks, triggering S-phase accumulation and apoptotic cell death. The potent cytotoxic activity of Zalypsis^® was ascertained in a 24 cell line panel. The mean IC₅₀ value was 7nM and leukemia and stomach tumor cell lines were amongst the most sensitive. Zalypsis^® administration in four murine xenograft models of human cancer demonstrates significant tumor growth inhibition that is highest in the Hs746t gastric cancer cell line with no weight loss of treated animals. Taken together, these results indicate that the potent antitumor activity of Zalypsis^® supports its current development in the clinic as an anticancer agent.

Keywords: Antineoplastic agents; Tetrahydroisoquinolines; DNA breaks; Apoptosis

Anti-inflammatory potential of 2-styrylchromones regarding their interference with arachidonic acid metabolic pathways by Ana Gomes; Eduarda Fernandes; Artur M.S. Silva; Diana C.G.A. Pinto; Clementina M.M. Santos; José A.S. Cavaleiro; José L.F.C. Lima (pp. 171-177).

Cyclooxygenases (COXs) are the key enzymes in the biosynthesis of prostanoids. COX-1 is a constitutive enzyme while the expression of COX-2 is highly stimulated in the event of inflammatory processes, leading to the production of large amounts of prostaglandins (PGs), in particular PGE₂ and PGI₂, which are pro-inflammatory mediators.Lipoxygenases (LOXs) are enzymes that produce hydroxy acids and leukotrienes (LTs). 5-LOX metabolizes arachidonic acid to yield, among other products, LTB₄, a potent chemoattractant mediator of inflammation.The aim of the present work was to evaluate the anti-inflammatory potential of 2-styrylchromones (2-SC), a chemical family of oxygen heterocyclic compounds, vinylogues of flavones (2-phenylchromones), by studying their COX-1 and COX-2 inhibitory capacity as well as their effects on the LTB₄ production by stimulated human polymorphonuclear leukocytes (PMNL).Some of the tested 2-SC were able to inhibit both COX-1 activity and LTB₄ production which makes them dual inhibitors of the COX and 5-LOX pathways. The most effective compounds in this study were those having structural moieties with proved antioxidant activity (3′,4′-catechol and 4′-phenol substituted B-rings).This type of compounds may exhibit anti-inflammatory activity with a wider spectrum than that of classical non-steroidal anti-inflammatory drugs (NSAIDs) by inhibiting 5-LOX product-mediated inflammatory reactions, towards which NSAIDs are ineffective.

Keywords: 2-Styrylchromones; Cyclooxygenase; 5-Lipoxigenase; Leukotriene B; ₄; Dual inhibitors; Inflammation

Curcumin attenuates 6-hydroxydopamine-induced cytotoxicity by anti-oxidation and nuclear factor-kappaB modulation in MES23.5 cells by Jun Wang; Xi-Xun Du; Hong Jiang; Jun-Xia Xie (pp. 178-183).

Oxidative stress has been implicated in the degeneration of dopaminergic neurons in the substantia nigra of Parkinson's disease patients, and several anti-oxidants have been shown to be effective on the treatment of Parkinson's disease. Curcumin has been previously reported to possess radical scavenger, iron chelating, anti-inflammatory properties in different tissues. The aim of present study is to explore the cytoprotection of curcumin against 6-hydroxydopamine (6-OHDA)-induced neuronal death, as well as the underlying mechanisms in MES23.5 cells. Our results showed that 6-OHDA significantly reduced the cell viability of MES23.5 cells. Curcumin protected MES23.5 cells against 6-OHDA neurotoxicity by partially restoring the mitochondrial membrane potential, increasing the level of Cu–Zn superoxide dismutase and suppressing an increase in intracellular reactive oxygen species. Furthermore, curcumin pretreatment significantly inhibited 6-OHDA induced nuclear factor-kappaB translocation. These results suggest that the neuroprotective effects of curcumin are attributed to the antioxidative properties and the modulation of nuclear factor-kappaB translocation.

Keywords: Oxidative stress; Parkinson's disease; Curcumin; 6-Hydroxydopamine; Nuclear factor-kappaB

Human hepatic CYP2B6 developmental expression: The impact of age and genotype by Edward L. Croom; Jeffrey C. Stevens; Ronald N. Hines; Andrew D. Wallace; Ernest Hodgson (pp. 184-190).

The percentage of pediatric liver samples with detectable CYP2B6 increased with age. Median CYP2B6 levels were higher in >30 days postnatal age samples compared to <30 days postnatal age samples.Although CYP2B6 is known to metabolize numerous pharmaceuticals and toxicants in adults, little is known regarding CYP2B6 ontogeny or its possible role in pediatric drug/toxicant metabolism. To address this knowledge gap, hepatic CYP2B6 protein levels were characterized in microsomal protein preparations isolated from a pediatric liver bank ( N=217). Donor ages ranged from 10 weeks gestation to 17 years of age with a median age of 1.9 months. CYP2B6 levels were measured by semi-quantitative western blotting. Overall, CYP2B6 expression was detected in 75% of samples. However, the percentage of samples with detectable CYP2B6 protein increased with age from 64% in fetal samples to 95% in samples from donors >10 years of age. There was a significant, but only 2-fold increase in median CYP2B6 expression after the neonatal period (birth to 30 days postnatal) although protein levels varied over 25-fold in both age groups. The median CYP2B6 level in samples over 30 postnatal days to 17 years of age (1.3pmol/mg microsomal protein) was lower than previously reported adult levels (2.2–22pmol/mg microsomal protein), however, this likely relates to the median age of these samples, i.e., 10.3 months. CYP2B6 expression did not vary significantly by gender. Furthermore, CYP2B6 levels did not correlate with CYP3A4, CYP3A5.1 or CYP3A7 activity, consistent with different mechanisms controlling the ontogeny and constitutive expression of these enzymes and the lack of significant induction in the pediatric samples.

Keywords: CYP450; Ontogeny; CYP2B6; CYP3A; Hepatic

Plasma membrane depolarization and Na,K-ATPase impairment induced by mitochondrial toxins augment leukemia cell apoptosis via a novel mitochondrial amplification mechanism by Wu Yin; Xiang Li; Su Feng; Wei Cheng; Bo Tang; Yi-Lin Shi; Zi-Chun Hua (pp. 191-202).

Na,K-ATPase is a ubiquitous transmembrane protein that regulates and maintains the intracellular Na⁺ and K⁺ gradient necessary for cell homeostasis. Recently, the importance of this pump in external stimuli-induced leukemia cell apoptosis has been increasingly appreciated, however, the exact role of Na,K-ATPase in mitochondrial apoptotic pathway still remains little understood. In this study, we found mitochondrial toxin rotenone caused a rapid mitochondrial membrane potential (MMP) collapse in Jurkat cells followed by plasma membrane depolarization (PMP). Similar results were also obtained in human U937 cells and non-cancerous mouse primary T cells. Rotenone-induced PMP depolarization occurred before apoptosis and well correlated with Na,K-ATPase impairment. To understand the mechanisms, Jurkat cells with mtDNA depletion and catalase overexpression were used. The results demonstrated that both PMP depolarization and Na,K-ATPase impairment induced by rotenone were regulated by mitochondrial H₂O₂ and Bcl-2. Finally, Na,K-ATPase suppression by ouabain greatly accelerated and enhanced mitochondrial toxins-induced cells apoptosis in Jurkat, U937 and primary T cells. In sum, by using leukemia cells and mouse primary T cells, we confirmed that mitochondria-to-Na,K-ATPase and PMP depolarization might represent a novel mechanism for mitochondria to amplify death signals in the initiation stage of cells apoptosis induced by mitochondrial toxins.

Keywords: Mitochondria; Na,K-ATPase; Depolarization; Reactive oxygen species; Apoptosis

Biochemistry, pharmacokinetics, and toxicology of a potent and selective DPP8/9 inhibitor by Jia-Jing Wu; Hung-Kuan Tang; Teng-Kuang Yeh; Chi-Min Chen; Hrong-Shing Shy; Yue-Ru Chu; Chia-Hui Chien; Ting-Yueh Tsai; Yu-Chen Huang; Yu-Lin Huang; Chih-Hsiang Huang; Huan-Yi Tseng; Weir-Torn Jiaang; Yu-Sheng Chao; Xin Chen (pp. 203-210).

No animal toxicities observed in a two-week study with Sprague–Dawley rats using a DPP8/9 inhibitor, which has high potency and selectivity, good membrane penetration and adequate tissue distribution.DPP-IV (EC 3.4.14.5) is a validated drug target for human type II diabetes. DPP-IV inhibitors without DPP8/9 inhibitory activity have been sought because a possible association has been reported between a “DPP8/9 inhibitor” and severe toxicity in animals. However, at present, it is not known whether the observed toxicity is associated with DPP8/9 inhibition, or an off-target effect induced by the compound. We investigated whether the inhibition of DPP8/9 is the cause of the severe toxicity in animals using a very potent and selective DPP8/9 inhibitor with different pharmacophore, 1G244. By Ki measurement, 1G244 is 15- and 8-fold more potent against DPP8 and DPP9, respectively, than the “DPP8/9 inhibitor”. Strikingly, the “DPP8/9 inhibitor” does not penetrate the plasma membrane but remains outside the cells, whereas 1G244 readily enters the cells, even at low doses. By repeatedly exposing Sprague–Dawley rats to 1G244 by intravenous injection for a period of 14 days, we observed no significant toxicological symptoms associated with 1G244. Blood and serum chemistry parameters were all within the normal ranges for the treated animals. Because of the high potency, good membrane penetration and adequate tissue distribution of 1G244, the mild symptoms observed are probably associated with DPP8/9 inhibition.

Keywords: Abbreviations; DPP; dipeptidyl peptidase; DPP-IV; dipeptidyl peptidase IV; FAP; fibroblast activation protein; FDA; the US Food and Drug Administration; IV; intravenous; SD; Sprague–Dawley; GLP; Good Laboratory Practice; CC; ₅₀; cytotoxic concentration; AUC; the area under the curveDipeptidyl peptidase; DPP8; DPP9; Inhibitor; Pharmacokinetics; Toxicity

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Biochemical Pharmacology (v.78, #2)