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Biochemical Pharmacology (v.81, #5)

Editorial Advisory Board (pp. iii).

Organic cation transporter OCT/SLC22A and H⁺/organic cation antiporter MATE/SLC47A are key molecules for nephrotoxicity of platinum agents by Atsushi Yonezawa; Ken-ichi Inui (pp. 563-568).

OCT2 is the determinant of cisplatin-induced nephrotoxicity. Cisplatin and oxaliplatin are substrates for OCT and MATE, but carboplatin and nedaplatin are not. Substrate specificity regulates the features of platinum agents.Platinum agents have been widely used in cancer chemotherapy for a long time. Cisplatin, carboplatin, oxaliplatin and nedaplatin have a common chemical structure consisting of platinum, carrier groups and leaving groups, and undergo the similar mechanism of cytotoxicity. However, each agent differs in its efficacy and adverse effects, although the molecular mechanism involved is unclear. Recently, it was reported that organic cation transporter OCT/SLC22A, and multidrug and toxin extrusion MATE/SLC47A play a role in the pharmacokinetics of platinum agents. Only cisplatin induces nephrotoxicity and the toxicity is kidney-specific. Kidney-specific OCT2 mediates the transport of cisplatin and is the determinant of cisplatin-induced nephrotoxicity. In addition, cisplatin and oxaliplatin are substrates for these transporters, but carboplatin and nedaplatin are not. Substrate specificity could regulate the features of platinum agents. In this commentary, we will discuss the characteristics of OCT and MATE, and demonstrate the recent topics about the relationship between the transport of platinum agents by organic cation transporters and their pharmacological characteristics.

Keywords: Abbreviations; OCT; organic cation transporter; MATE; multidrug and toxin extrusion; Ctr1; copper transporter 1; DACH; diaminocyclohexaneCisplatin; Renal handling; Pharmacokinetics; Drug transporter; Copper transporter Ctr1

Sirtuins and inflammation: Friends or foes? by Mara Gallí; Frédéric Van Gool; Oberdan Leo (pp. 569-576).

Lysine acetylation/deacetylation has been recognized as an important posttranslational modification regulating numerous cellular processes. Sirtuins represent novel players in these complex regulatory circuits. These NAD-dependent lysine-deacetylases have attracted much interest based on their role in the regulation of lifespan in lower organisms, and their capacity to interfere with cell growth, proliferation and survival in response to stress. Their absolute requirement for NAD suggests that these enzymes may represent an important molecular link between metabolism and several human disorders such as diabetes and cancer. More recently, the identification of several transcription factors known to play a role in the immune system as sirtuin substrates has suggested that this family of enzymes may also play an important role in the regulation of inflammation, a pathological situation with clear links to metabolism and aging in humans. We review herein the possible links between nuclear sirtuins and the regulation of an immune response, and discuss the possible strategies that may lead to the development of novel therapeutic approaches to treat inflammation by targeting sirtuin activity.

Nephroblastoma overexpressed gene (NOV) enhances cell motility and COX-2 upregulation of human osteosarcoma involves αvβ5 integrin, ILK and AP-1-dependent pathways by Chun-Yin Huang; Chun-Yi Lee; Meng-Yi Chen; Hsiao-Chi Tsai; Horng-Chaung Hsu; Chih-Hsin Tang (pp. 577-585).

Osteosarcoma is characterized by a high malignant and metastatic potential. Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin synthase, has been implicated in tumor metastasis. Nephroblastoma overexpressed gene (NOV), also called CCN3, was regulated proliferation and differentiation of cancer cells. However, the effect of NOV on migration activity and COX-2 expression in human osteosarcoma cells is mostly unknown. Here we found that NOV increased the migration and expression of COX-2 in human osteosarcoma cells. αvβ5 monoclonal antibody (mAb), integrin-linked kinase (ILK) and Akt inhibitor reduced the NOV-enhanced the migration and COX-2 up-regulation of osteosarcoma cells. NOV stimulation increased the ILK kinase activity and phosphorylation of Akt. In addition, c-Jun siRNA also antagonized the NOV-mediated migration and COX-2 expression. Moreover, NOV enhanced the AP-1 binding activity and promoter activity. Taken together, these results suggest that the NOV acts through αvβ5 integrin to activate ILK and Akt, which in turn activates c-Jun and AP-1, resulting in the activations of COX-2 and contributing the migration of human osteosarcoma cells.

Keywords: NOV; Integrin; Osteosarcoma; Migration; ILK

Nucleoside analogs induce proteasomal down-regulation of p21 in chronic lymphocytic leukemia cell lines by L. Bastin-Coyette; S. Cardoen; C. Smal; E. de Viron; A. Arts; R. Amsailale; E. Van Den Neste; F. Bontemps (pp. 586-593).

2-Chorodeoxyadenosirne (CdA) down-regulates p21 despite p53 activation in CLL cell lines. Cdk2 activation and PCNA monoubiquitination, which are known to be negatively regulated by p21, are also induced. These events constitute an alternate pattern of cell response to nucleoside analogs.Nucleoside analogs (NAs) represent an important class of anticancer agents that induce cell death after conversion to triphosphate derivatives. One of their most important mechanisms of action is the activation of p53, leading to apoptosis through the intrinsic pathway. Classically, the activation of p53 also induces p21 accumulation, which leads to cell cycle arrest at the G1/S transition. In previous work, we observed that 2-chloro-2′-deoxyadenosine (CdA), a NA with high activity in lymphoid disorders, including chronic lymphocytic leukemia (CLL), promotes the G1/S transition in the CLL cell line EHEB at cytotoxic concentrations. This finding led us to investigate the p21 response to NAs in these cells. We show here that CdA, but also fludarabine, gemcitabine, and cytarabine, strongly reduced the p21 protein level in EHEB cells as well as in JVM-2 cells, another CLL cell line. This p21 depletion occurred despite induction of p53 and increase of p21 mRNA and was prevented by proteasome inhibitors. Increase of proteasomal degradation caused by NAs appeared to be ubiquitin-independent. Also, NAs induced in these cells an increase of cyclin-dependent kinase (Cdk2) activity and a monoubiquitination of cell proliferating nuclear antigen (PCNA), two processes that are negatively regulated by p21. These changes were not observed with other p53 activators, like etoposide and nutlin-3a that increased the p21 protein level. In conclusion, our study reveals that NAs can induce an alternative pattern of cellular response in some cell models.

Keywords: p53; p21 depletion; Nucleoside analogs; PCNA monoubiquitination; Cdk2; CLL

Autophagy inhibition enhances apoptosis triggered by BO-1051, an N-mustard derivative, and involves the ATM signaling pathway by Li-Hsin Chen; Che-Chuan Loong; Tsann-Long Su; Yi-Jang Lee; Pei-Ming Chu; Ming-Long Tsai; Ping-Hsin Tsai; Pang-Hsien Tu; Chin-Wen Chi; Hsin-Chen Lee; Shih-Hwa Chiou (pp. 594-605).

In a previous study, BO-1051, an N-mustard linked with a DNA-affinic molecule, was shown to target various types of cancer cell lines. In the present study, we aimed to investigate the cytotoxicity, as well as the underlying mechanism, of BO-1051. We found that BO-1051 simultaneously induced apoptosis and autophagy in hepatocellular carcinoma cell lines. DNA double strand breaks induced by BO-1051 activated the ATM signaling pathway and subsequently resulted in caspase-dependent apoptosis. When autophagy was inhibited in its early or late stages, apoptosis was significantly enhanced. This result indicated autophagy as a cytoprotective effect against BO-1051-induced cell death. We further inhibited ATM activation using an ATM kinase inhibitor or ATM-specific siRNA and found that while apoptosis was blocked, autophagy also diminished in response to BO-1051. We not only determined a signaling pathway induced by BO-1051 but also clarified the linkage between DNA damage-induced apoptosis and autophagy. We also showed that BO-1051-induced autophagy acts as a cytoprotective reaction and downstream target of the ATM-signaling pathway. This research revealed autophagy as a universal cytoprotective response against DNA damage-inducing chemotherapeutic agents, including BO-1051, cisplatin, and doxorubicin, in hepatocellular carcinoma cell lines. Autophagy contributes to the remarkable drug resistance ability of liver cancer.

Keywords: N-mustard; Apoptosis; Autophagy; ATM; Hepatoma

NF-kappaB inhibition improves the sensitivity of human glioblastoma cells to 5-aminolevulinic acid-based photodynamic therapy by Isabelle Coupienne; Sébastien Bontems; Michael Dewaele; Noemi Rubio; Yvette Habraken; Simone Fulda; Patrizia Agostinis; Jacques Piette (pp. 606-616).

Glioblastoma constitute the most frequent and deadliest brain tumors of astrocytic origin. They are very resistant to all current therapies and are associated with a huge rate of recurrence. In most cases, this type of tumor is characterized by a constitutive activation of the nuclear factor-kappaB (NF-κB). This factor is known to be a key regulator of various physiological processes such as inflammation, immune response, cell growth or apoptosis. In the present study, we explored the role of NF-κB activation in the sensitivity of human glioblastoma cells to a treatment by 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT). 5-ALA is a physiological compound widely used in PDT as well as in tumor photodetection (PDD). Our results show that inhibition of NF-κB improves glioblastoma cell death in response to 5-ALA-PDT. We then studied the molecular mechanisms underlying the cell death induced by PDT combined or not with NF-κB inhibition. We found that apoptosis was induced by PDT but in an incomplete manner and that, unexpectedly, NF-κB inhibition reduced its level. Oppositely PDT mainly induces necrosis in glioblastoma cells and NF-κB is found to have anti-necrotic functions in this context. The autophagic flux was also enhanced as a result of 5-ALA-PDT and we demonstrate that stimulation of autophagy acts as a pro-survival mechanism confering protection against PDT-mediated necrosis. These data point out that 5-ALA-PDT has an interesting potential as a mean to treat glioblastoma and that inhibition of NF-κB renders glioblastoma cells more sensitive to the treatment.

Keywords: Abbreviations; 5-ALA; 5-aminolevulinic acid; BCL2; B-cell lymphoma 2; c-FLIP; cellular FLICE-inhibitory protein; EMSA; electrophoretic mobility shift assay; IAP; inhbitor of apoptosis; IκB; inhibitory κB; LDH; lactate dehydrogenase; LTR; long terminal repeat; mTOR; mammalian target of rapamycin; NF-κB; nuclear factor kappa B; PDD; photodiagnosis; PDT; photodynamic therapy; PI; propidium iodide; PPIX; protoporphyrin IX; ROS; reactive oxygen species; SR; IκBα super-repressor; Tunel; terminal deoxynucleotidyl transferase dUTP nick end labeling; XIAP; X-linked inhibitor of apoptosisKey words; Photodynamic therapy; NF-κB; Glioblastoma; Necrosis; Autophagy; Apoptosis

Cis-4-methylsphingosine is a sphingosine-1-phosphate receptor modulator by Michael ter Braak; Ralf Frederik Claas; Bianca Hegen; Sandra Labocha; Nerea Ferreirós; Josef Pfeilschifter; Andrea Huwiler; Gerhild van Echten-Deckert; Dagmar Meyer zu Heringdorf (pp. 617-625).

Sphingosine-1-phosphate (S1P) acts as high affinity agonist at specific G-protein-coupled receptors, S1P_1–5, that play important roles e.g. in the cardiovascular and immune systems. A S1P receptor modulating drug, FTY720 (fingolimod), has been effective in phase III clinical trials for multiple sclerosis. FTY720 is a sphingosine analogue and prodrug of FTY720-phosphate, which activates all S1P receptors except S1P₂ and disrupts lymphocyte trafficking by internalizing the S1P₁ receptor. Cis-4-methylsphingosine (cis-4M-Sph) is another synthetic sphingosine analogue that is readily taken up by cells and phosphorylated to cis-4-methylsphingosine-1-phosphate (cis-4M-S1P). Therefore, we analysed whether cis-4M-Sph interacted with S1P receptors through its metabolite cis-4M-S1P in a manner similar to FTY720. Indeed, cis-4M-Sph caused an internalization of S1P receptors, but differed from FTY720 as it acted on S1P₂ and S1P₃ and only weakly on S1P₁, while FTY720 internalized S1P₁ and S1P₃ but not S1P₂. Consequently, pre-incubation with cis-4M-Sph specifically desensitized S1P-induced [Ca²⁺]_i increases, which are mediated by S1P₂ and S1P₃, in a time- and concentration-dependent manner. This effect was not shared by sphingosine or FTY720, indicating that metabolic stability and targeting of S1P₂ receptors were important. The desensitization of S1P-induced [Ca²⁺]_i increases was dependent on the expression of SphKs, predominantly of SphK2, and thus mediated by cis-4M-S1P. In agreement, cis-4M-S1P was detected in the supernatants of cells exposed to cis-4M-Sph. It is concluded that cis-4M-Sph, through its metabolite cis-4M-S1P, acts as a S1P receptor modulator and causes S1P receptor internalization and desensitization. The data furthermore help to define requirements for sphingosine kinase substrates as S1P receptor modulating prodrugs.

Keywords: Abbreviations; BSA; bovine serum albumin; [Ca; ²⁺; ]; _i; intracellular free Ca; ²⁺; concentration; Cis-4M-S1P; cis-4-methylsphingosine-1-phosphate; Cis-4M-Sph; cis-4-methylsphingosine; LC–MS/MS; liquid chromatography–tandem mass spectrometry; LPA; lysophosphatidic acid; MEFs; mouse embryonic fibroblasts; PTX; pertussis toxin; S1P; sphingosine-1-phosphateSphingosine-1-phosphate; Lysophospholipid receptors; GPCR; Sphingosine kinase; FTY720

Inhibition of human leukocyte elastase, plasmin and matrix metalloproteinases by oleic acid and oleoyl-galardin derivative(s) by Gautier Moroy; Erika Bourguet; Martine Decarme; Janos Sapi; Alain J.P. Alix; William Hornebeck; Sandrine Lorimier (pp. 626-635).

Incorporation of oleic acid at P′2 position of Galardin^® leads to MMP-13 selectivity. Besides, this compound inhibits neutrophil elastase and plasmin activity.Molecular modeling was undertaken at aims to analyze the interactions between oleic acid and human leukocyte elastase (HLE), plasmin and matrix metalloproteinase-2 (MMP-2), involved in the inhibitory capacity of fatty acid towards those proteases. The carboxylic acid group of the fatty acid was found to form a salt bridge with Arg²¹⁷ of HLE while unsaturation interacted with Phe¹⁹² and Val²¹⁶ at the S₃ subsite, and alkyl end group occupied S₁ subsite. In keeping with the main contribution of kringle 5 domain in plasmin–oleic acid interaction [Huet E et al. Biochem Pharmacol 2004;67(4):643–54], docking computations revealed that the long alkyl chain of fatty acid inserted within an hydrophobic groove of this domain with the carboxylate forming a salt bridge with Arg⁵¹². Finally, blind docking revealed that oleic acid could occupy both S′₁ subsite and Fn(II)₃ domain of MMP-2. Several residues involved in Fn(II)₃/oleic acid interaction were similarly implicated in binding of this domain to collagen.Oleic acid was covalently linked to galardin (at P′₂ position): OL-GAL (CONHOH) or to its carboxylic acid counterpart: OL-GAL (COOH), with the idea to obtain potent MMP inhibitors able to also interfere with elastase and plasmin activity. OL-GALs were found less potent MMP inhibitors as compared to galardin and no selectivity for MMP-2 or MMP-9 could be demonstrated. Docking computations indicated that contrary to oleic acid, OL-GAL binds only to MMP-2 active site and surprisingly, hydroxamic acid was unable to chelate Zn, but instead forms a salt bridge with the N-terminal Tyr¹¹⁰. Interestingly, oleic acid and particularly OL-GALs proved to potently inhibit MMP-13. OL-GAL was found as potent as galardin ( K_i equal to 1.8nM for OL-GAL and 1.45nM for GAL) and selectivity for that MMP was attained (2–3 log orders of difference in inhibitory potency as compared to other MMPs).Molecular modeling studies indicated that oleic acid could be accommodated within S′₁ pocket of MMP-13 with carboxylic acid chelating Zn ion. OL-GAL also occupied such pocket but hydroxamic acid did not interact with Zn but instead was located at 2.8Å from Tyr¹⁷⁶.Since these derivatives retained, as their oleic acid original counterpart, the capacity to inhibit the amidolytic activity of HLE and plasmin as well as to decrease HLE- and plasmin-mediated pro MMP-3 activation, they might be of therapeutic value to control proteolytic cascades in chronic inflammatory disorders.

Keywords: Abbreviations; HLE; human leukocyte elastase; Fn; fibronectin domain; MMP; matrix metalloproteinase; OL-GAL; oleoyl-galardin derivativesLeukocyte elastase; Plasmin; Matrix metalloproteinase; Oleic acid; Oleoyl-galardin; Molecular docking

Effect of apocynin, a NADPH oxidase inhibitor, on acute lung inflammation by Daniela Impellizzeri; Emanuela Esposito; Emanuela Mazzon; Irene Paterniti; Rosanna Di Paola; Placido Bramanti; Salvatore Cuzzocrea (pp. 636-648).

NADPH-oxidase is an enzyme responsible for reactive oxygen species production (ROS) and inhibition of this enzyme represents an attractive therapeutic target for the treatment of many diseases. The aim of this study was to investigate the effects of apocynin, a NADPH-oxidase inhibitor, in a mouse model of carrageenan-induced pleurisy. Injection of carrageenan into the pleural cavity of mice elicited an acute inflammatory response characterized by: infiltration of neutrophils in lung tissues and subsequent lipid peroxidation, increased production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and increased expression of intercellular adhesion molecule (ICAM-1) and platelet-adhesion molecule (P-selectin). Furthermore, carrageenan induced the expression of nuclear factor-κB (NF-κB) inducible nitric oxide synthase (iNOS), nitrotyrosine, poly-ADP-ribosyl polymerase (PARP) as well as induced apoptosis (TUNEL staining, FAS-ligand expression, Bax and Bcl-2 expression) and mitogen-activated protein kinase (MAPK) activation in the lung tissues. Administration of apocynin, 30min after the challenge with carrageenan, caused a significant reduction of all the parameters of inflammation measured. Thus, based on these findings we propose that NADPH oxidase inhibitor such as apocynin may be useful in the treatment of various inflammatory diseases.

Keywords: Carrageenan; Pleurisy; Neutrophils; Oxidative stress; Cytokines; Apoptosis

Soraphen A, an inhibitor of acetyl CoA carboxylase activity, interferes with fatty acid elongation by Donald B. Jump; Moises Torres-Gonzalez; L. Karl Olson (pp. 649-660).

Acetyl CoA carboxylase (ACC1 and ACC2) generates malonyl CoA, a substrate for de novo lipogenesis (DNL) and an inhibitor of mitochondrial fatty acid β-oxidation (FAO). Malonyl CoA is also a substrate for microsomal fatty acid elongation, an important pathway for saturated (SFA), mono- (MUFA) and polyunsaturated fatty acid (PUFA) synthesis. Despite the interest in ACC as a target for obesity and cancer therapy, little attention has been given to the role ACC plays in long chain fatty acid synthesis. This report examines the effect of pharmacological inhibition of ACC on DNL and palmitate (16:0) and linoleate (18:2, n−6) metabolism in HepG2 and LnCap cells. The ACC inhibitor, soraphen A, lowers cellular malonyl CoA, attenuates DNL and the formation of fatty acid elongation products derived from exogenous fatty acids, i.e., 16:0 and 18:2, n−6; IC₅₀∼5nM. Elevated expression of fatty acid elongases (Elovl5, Elovl6) or desaturases (FADS1, FADS2) failed to override the soraphen A effect on SFA, MUFA or PUFA synthesis. Inhibition of fatty acid elongation leads to the accumulation of 16- and 18-carbon unsaturated fatty acids derived from 16:0 and 18:2, n−6, respectively. Pharmacological inhibition of ACC activity will not only attenuate DNL and induce FAO, but will also attenuate the synthesis of very long chain saturated, mono- and polyunsaturated fatty acids.

Keywords: Abbreviations; ACC; acetyl CoA carboxylase; ASM; acid soluble material; ARA; arachidonic acid (20:4,; n; −; 6); CPT1; carnitine palmitoyl transferase 1; DHA; docosahexaenoic acid (22:6,; n; −; 3); DNL; de novo lipogenesis; FADS1; Δ; ⁵; -desaturase; FADS2; Δ; ⁶; -desaturase; DHGL; dihomo-γ-linolenic acid (20:3,; n; −; 6); Elovl; fatty acid elongase; FASN; fatty acid synthase; LUC; luciferase; MCD; malonyl CoA decarboxylase; MUFA; monounsaturated fatty acid; NEFA; non-esterified fatty acid; pβOx; peroxisomal β oxidation; PUFA; polyunsaturated fatty acid; RP-HPLC; reverse phase high performance chromatography; SFA; saturated fatty acid; SCD1; stearoyl CoA desaturase; RT-PCR; real time PCR; TLC; thin layer chromatographyAcetyl CoA carboxylase; Soraphen A; De novo lipogenesis; Fatty acid elongation; Fatty acid desaturation; Fatty acid oxidation

The N-terminal domain of human hemokinin-1 influences functional selectivity property for tachykinin receptor neurokinin-1 by Lingyun Mou; Yanhong Xing; Ziqing Kong; Ying Zhou; Zongyao Chen; Rui Wang (pp. 661-668).

Human hemokinin-1 (hHK-1) is a substance P-like tachykinin peptide preferentially expressed in non-neuronal tissues. It is involved in multiple physiological functions such as inflammation, hematopoietic cells development and vasodilatation via the interaction with tachykinin receptor neurokinin-1 (NK1). To further understand the intracellular signal transduction mechanism under such functional multiplicity, current study was focused on the differential activation of Gs and Gq pathways by hHK-1 and its C-terminal fragments, which is termed as functional selectivity. We demonstrated these hHK-1 and related peptide fragments can independently activate Gs and Gq pathways, showing a relative bias toward Gq over Gs pathway. The T1, K3 and Q6 of hHK-1 might play roles in the activation of adenylate cyclase mediated by Gs, while having negligible effect on Gq mediated intracellular calcium release. The stepwise truncation of N-terminal amino acid of hHK-1 caused gradual decrease in ERK1/2 phosphorylation level and NF-κB activity. However, it had little influence on the induction of NK1 receptor desensitization and internalization. Taken together these data support that hHK-1 and its C-terminal fragments are human NK1 receptor agonists with different functional selectivity properties and that such functional selectivity leads to differential activation of downstream signaling and receptor trafficking.

Keywords: Abbreviations; cAMP; cyclic adenosine monophosphate; NF-κB; nuclear factor kappa B; ERK1/2; extracellular regulated protein kinases1/2Hemokinin-1; Functional selectivity; Neurokinin-1 receptor; ERK1/2; NF-κB

Evaluation of in vitro PXR-based assays and in silico modeling approaches for understanding the binding of a structurally diverse set of drugs to PXR by Li Xiao; Elliott Nickbarg; Wenyan Wang; Ann Thomas; Michael Ziebell; Winfred W. Prosise; Charles A. Lesburg; S. Shane Taremi; Valerie L. Gerlach; Hung V. Le; K.-C. Cheng (pp. 669-679).

3D structure of a tethered PXR–SRC peptide/SR12813.The pregnane X-receptor (PXR) is a promiscuous nuclear receptor primarily responsible for the induction of genes from the cytochrome P450 3A family. In this study, we used a previously described PXR/SRC tethered protein to establish two in vitro assays for identifying PXR ligands: automated ligand identification system (ALIS) and temperature-dependent circular dichroism (TdCD). Kd values determined by ALIS and TdCD showed good correlations with the EC50 values determined by a PXR luciferase reporter-gene assay for 37 marketed drugs. The same set of compounds was modeled into the PXR ligand-binding domain that takes into consideration the structural variations of five published X-ray structures of PXR–ligand complexes. Major findings from our in silico analysis are as follows. First, the primary determinants for non-binders of PXR are molecular size and shape of the compounds. Low molecular weight (MW<300) compounds were in general found to be non-binders, and those molecules that do not match the shape of the PXR ligand-binding site may also act as a non-binder. Secondly, the favorable hydrophobic interactions, mostly through aromatic π–π interactions, and the presence of suitable hydrogen bond(s) between the compounds and PXR are attributes of strong binders. Thirdly, the structures of the PXR binding domain possess the flexibility that accommodates structurally diverse compounds, while some of the strong binders may also adapt flexible conformations for fitting into the binding site. The results from this study provide a molecular basis for future efforts in reducing/abolishing the PXR-dependent CYP3A4 induction liability.

Keywords: Abbreviations; PXR; pregnane X receptor; CYP; cytochrome P450; XRE; xenobiotic response element; SRC; steroid receptor co-activator; ALIS; automated ligand identification system; TdCD; temperature-dependent circular dichroism; Kd; dissociation constant; RXR; retinoic X receptor; CAR; constitutive androstane receptor; VDR; vitamin D receptor; LBD; ligand-binding domain; MW; molecular weight; Da; Dalton; DDI; drug–drug interactionCYP3A induction; Pregnane X receptor; In vitro; assays

Modulation of Rad51, ERCC1, and thymidine phosphorylase by emodin result in synergistic cytotoxic effect in combination with capecitabine by Jen-Chung Ko; Min-Shao Tsai; Ya-Hsun Kuo; Yu-Fan Chiu; Shao-Hsing Weng; Ying-Chen Su; Yun-Wei Lin (pp. 680-690).

Thymidine phosphorylase (TP) is the rate-limiting enzyme for the activation of capecitabine (pro-drug of fluorouracil), and as a useful predictor of tumor response to capecitabine-based chemotherapy. Overexpression of Rad51 and ERCC1 induce resistance to chemotherapeutic agents. Emodin, one of the main bioactive anthraquinone derivatives in the roots and rhizomes of numerous plants, possesses potent antitumor effects. Accordingly, we aimed to explore the molecular mechanism of emodin enhances the capecitabine-induced cytotoxicity through controlling Rad51, ERCC1, and TP expression in human non-small cell lung cancer (NSCLC). The results show that capecitabine increases the phosphorylation of MKK1/2–ERK1/2 and protein levels of Rad51 and ERCC1 through enhancing the protein stability. Depletion of endogenous Rad51 or ERCC1 expression by specific small interfering RNA transfection significantly increases capecitabine-induced cell death and growth inhibition. Emodin enhances the capecitabine-induced cytotoxic effects through ERK1/2 inactivation and decreasing the Rad51 and ERCC1 protein levels induced by capecitabine. Enhancement of ERK1/2 signaling by constitutively active MKK1/2 (MKK1/2-CA) results in increasing Rad51 and ERCC1 protein levels and cell viability in NSCLC cell lines treated with emodin and capecitabine. Interestingly, emodin enhances TP mRNA and protein expression in capecitabine treated NSCLC cell lines, and depletion of the TP expression decreases the cytotoxic effects induced by capecitabine and emodin. We conclude that enhancing the cytotoxicity to capecitabine by emodin is mediated by down-regulation the expression of Rad51 and ERCC1 and up-regulation TP expression.

Keywords: ERCC1; Rad51; Thymidine phosphorylase; Capecitabine; Emodin; ERK1/2; Cytotoxicity; Non-small cell lung cancer

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Biochemical Pharmacology (v.81, #5)