Reactive metabolite of trovafloxacin activates inflammasomes: Implications for trovafloxacin-induced liver injury.

Trovafloxacin is a quinolone antibiotic drug with broad-spectrum activity, which was withdrawn from a global market relatively soon after approval because of serious liver injury. The characteristics of trovafloxacin-induced liver injury are consistent with an idiosyncratic reaction; however, the details of the mechanism have not been elucidated. We examined whether trovafloxacin induces the release of damage-associated molecular patterns (DAMPs) that activate inflammasomes. We also tested ciprofloxacin, levofloxacin, gatifloxacin, and grepafloxacin for their ability to activate inflammasomes. Drug bioactivation was performed with human hepatocarcinoma functional liver cell-4 (FLC-4) cells, and THP-1 cells (human monocyte cell line) were used for the detection of inflammasome activation. The supernatant from the incubation of trovafloxacin with FLC-4 cells for 7 days increased caspase-1 activity and production of IL-1ß by THP-1 cells. In the supernatant of FLC-4 cells that had been incubated with trovafloxacin, heat shock protein (HSP) 40 was significantly increased. Addition of a cytochrome P450 inhibitor to the FLC-4 cells prevented the release of HSP40 from the FLC-4 cells and inflammasome activation in THP-1 cells by the FLC-4 supernatant. These results suggest that reactive metabolites of trovafloxacin can cause the release of DAMPs from hepatocytes that can activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by trovafloxacin, which, in some patients, can cause immune-related liver injury.

Trovafloxacin drives inflammation-associated drug-induced adverse hepatic reaction by changing macrophage polarization.

Drug-induced liver injury (DILI) is an adverse hepatic reaction and a serious concern for public healthcare systems and pharmaceutical companies. DILI is frequently caused by a combination of direct toxic stresses and subsequent immune damage to hepatocytes. However, little is known about the mechanism by which drugs facilitate the activation of the innate immune system. Here, we aimed to decipher the inflammatory events in trovafloxacin (TVX)-induced reactions using liver macrophages. We showed that proinflammatory M1-like macrophages mainly contributed to hepatotoxicity mediated by TVX, a DILI drug. Additionally, transcriptome results showed that the interferon type I pathway, cytokines, and apoptosis pathway were involved in the initiation of synergistic effects resulting in TVX-induced liver injury. We hypothesized that DILI drugs could drive liver injury by altering the activation and phenotype of hepatic macrophages. Furthermore, drug treatment-induced transcriptional changes such as Traf1 and 2, Socs3, and Hbegf in macrophage polarization could be used to assess drug-specific immune-mediated reactions. Therefore, we proposed that transcriptional change in the genes related to macrophage polarization index could be an indicator to reflect the severity of DILI in a preclinical setting during drug development.

TNFα enhances trovafloxacin-induced in vitro hepatotoxicity by inhibiting protective autophagy.

Trovafloxacin (TVX) is associated with idiosyncratic drug-induced liver injury (iDILI) and inflammation-mediated hepatotoxicity. However, the inflammatory stress-regulated mechanisms in iDILI remain unclear. Herein, we elucidated the novel role of tumor-necrosis factor alpha (TNFα), an inflammatory stress factor, in TVX-induced in vitro hepatotoxicity and synergistic toxicity. TVX specifically induced synergistic toxicity in HepG2 cells with TNFα, which inhibits autophagy. TVX-treated HepG2 cells induced protective autophagy by inhibiting the expression of mTOR signaling proteins, while ATG5 knockdown in HepG2 cells, responsible for the impairment of autophagy, enhanced TVX-induced toxicity due to the increase in cytochrome C release and JNK pathway activation. Interestingly, the expression of mTOR signal proteins, which were suppressed by TVX, disrupted the negative feedback of the PI3K/AKT pathway and TNFα rebounded p70S6K phosphorylation. Co-treatment with TVX and TNFα inhibited protective autophagy by maintaining p70S6K activity, which enhanced TVX-induced cytotoxicity. Phosphorylation of p70S6K was inhibited by siRNA knockdown and rapamycin to restore TNFα-inhibited autophagy, which prevented the synergistic effect on TVX-induced cytotoxicity. These results indicate that TVX activates protective autophagy in HepG2 cells exposed to toxicity and an imbalance in negative feedback regulation of autophagy by TNFα synergistically enhanced the toxicity. The finding from this study may contribute to a better understanding of the mechanisms underlying iDILI associated with inflammatory stress.

A Multimodal Genotoxic Anticancer Drug Characterized by Pharmacogenetic Analysis in Caenorhabditis elegans.

New anticancer therapeutics require extensive in vivo characterization to identify endogenous and exogenous factors affecting efficacy, to measure toxicity and mutagenicity, and to determine genotypes that result in therapeutic sensitivity or resistance. We used Caenorhabditis elegans as a platform with which to characterize properties of the anticancer therapeutic CX-5461. To understand the processes that respond to CX-5461-induced damage, we generated pharmacogenetic profiles for a panel of C. elegans DNA replication and repair mutants with common DNA-damaging agents for comparison with the profile of CX-5461. We found that multiple repair pathways, including homology-directed repair, microhomology-mediated end joining, nucleotide excision repair, and translesion synthesis, were needed for CX-5461 tolerance. To determine the frequency and spectrum of CX-5461-induced mutations, we used a genetic balancer to capture CX-5461-induced mutations. We found that CX-5461 is mutagenic, resulting in both large copy number variations and a high frequency of single-nucleotide variations (SNVs), which are consistent with the pharmacogenetic profile for CX-5461. Whole-genome sequencing of CX-5461-exposed animals found that CX-5461-induced SNVs exhibited a distinct mutational signature. We also phenocopied the CX-5461 photoreactivity observed in clinical trials and demonstrated that CX-5461 generates reactive oxygen species when exposed to UVA radiation. Together, the data from C. elegans demonstrate that CX-5461 is a multimodal DNA-damaging anticancer agent.

Old drug repurposing for neglected disease: Pyronaridine as a promising candidate for the treatment of Echinococcus granulosus infections.

BACKGROUND: Cystic echinococcosis (CE), a condition caused by the larval stage of the dog tapeworm Echinococcus granulosus sensu stricto, is a globally distributed zoonotic disease. Current treatment options for CE are limited, and an effective and safe anti-echinococcal drug is urgently required. METHODS: Drug repurposing strategy was employed to identify new therapeutic agents against echinococcal cysts. An in vitro protoscolicidal assay along with in vivo murine models was applied in the drug screening. A microinjection procedure was employed to mimic the clinical PAIR (puncture, aspiration, injection and reaspiration) technique to evaluate the potential application of the candidate drug in clinical practice. FINDINGS: We repurposed pyronaridine, an approved antimalarial drug, for the treatment of CE. Following a three-dose intraperitoneal regimen (57 mg/kg, q.d. for 3 days), pyronaridine caused 100% cyst mortality. Oral administration of pyronaridine at 57 mg/kg, q.d. for 30 days significantly reduced the parasitic burden in the pre-infected mice compared with albendazole group (p < 0.001). Using a microinjection of drug into cysts, pyronaridine (200 µM) showed highly effective in term of inhibition of cyst growth (p < 0.05, compared with saline group). Pharmacokinetic analysis revealed that pyronaridine was highly distributed in the liver and lungs, the most affected organs of CE. Function analysis showed that pyronaridine inhibited the activity of topoisomerase I (IC50 = 209.7 ± 1.1 µM). In addition, classical apoptotic hallmarks, including DNA fragmentation and caspase activation, were triggered. INTERPRETATION: Given its approved clinical safety, the repurposing of pyronaridine offers a rapidly translational option for treating CE including PAIR. FUND: National Natural Science Foundation of China and International Cooperation Project of the Qinghai Science and Technology Department.

The hepatotoxic fluoroquinolone trovafloxacin disturbs TNF- and LPS-induced p65 nuclear translocation in vivo and in vitro.

Idiosyncratic drug-induced liver injury (IDILI) is a severe disease that cannot be detected during drug development. It has been shown that hepatotoxicity of some compounds associated with IDILI becomes apparent when these are combined in vivo and in vitro with LPS or TNF. Among these compounds trovafloxacin (TVX) induced apoptosis in the liver and increased pro-inflammatory cytokines in mice exposed to LPS/TNF. The hepatocyte survival and the cytokine release after TNF/LPS stimulation relies on a pulsatile activation of NF-κB. We set out to evaluate the dynamic activation of NF-κB in response to TVX + TNF or LPS models, both in mouse and human cells. Remarkably, TVX prolonged the first translocation of NF-κB induced by TNF both in vivo and in vitro. The prolonged p65 translocation caused by TVX was associated with an increased phosphorylation of IKK and MAPKs and accumulation of inhibitors of NF-κB such as IκBα and A20 in HepG2. Coherently, TVX suppressed further TNF-induced NF-κB translocations in HepG2 leading to decreased transcription of ICAM-1 and inhibitors of apoptosis. TVX prolonged LPS-induced NF-κB translocation in RAW264.7 macrophages increasing the secretion of TNF. In summary, this study presents new, relevant insights into the mechanism of TVX-induced liver injury underlining the resemblance between mouse and human models. In this study we convincingly show that regularly used toxicity models provide a coherent view of relevant pathways for IDILI. We propose that assessment of the kinetics of activation of NF-κB and MAPKs is an appropriate tool for the identification of hepatotoxic compounds during drug development.

Synthesis and photocytotoxic activity of [1,2,3]triazolo[4,5-h][1,6]naphthyridines and [1,3]oxazolo[5,4-h][1,6]naphthyridines.

[1,2,3]Triazolo[4,5-h][1,6]naphthyridines and [1,3]oxazolo[5,4-h][1,6]naphthyridines were synthesized with the aim to investigate their photocytotoxic activity. Upon irradiation, oxazolo-naphtapyridines induced light-dependent cell death at nanomolar/low micromolar concentrations (EC50 0.01-6.59 µM). The most photocytotoxic derivative showed very high selectivity and photocytotoxicity indexes (SI = 72-86, PTI>5000), along with a triplet excited state with exceptionally long lifetime (18.0 µs) and high molar absorptivity (29781 ±â€¯180 M-1cm-1 at λmax 315 nm). The light-induced production of ROS promptly induced an unquenchable apoptotic process selectively in tumor cells, with mitochondrial and lysosomal involvement. Altogether, these results demonstrate that the most active compound acts as a promising singlet oxygen sensitizer for biological applications.

Trovafloxacin-Induced Liver Injury: Lack in Regulation of Inflammation by Inhibition of Nucleotide Release and Neutrophil Movement.

The fluoroquinolone trovafloxacin (TVX) is associated with a high risk of drug-induced liver injury (DILI). Although part of the liver damage by TVX+TNF relies on neutrophils, we have recently demonstrated that liver recruitment of monocytes and neutrophils is delayed by TVX. Here we show that the delayed leukocyte recruitment is caused by a combination of effects which are linked to the capacity of TVX to block the hemichannel pannexin 1. TVX inhibited find-me signal release in apoptotic HepG2 hepatocytes, decelerated freshly isolated human neutrophils toward IL-8 and f-MLF, and decreased the liver expression of ICAM-1. In blood of TVX+TNF-treated mice, we observed an accumulation of activated neutrophils despite an increased MIP-2 release by the liver. Depletion of monocytes and neutrophils caused increased serum concentrations of TNF, IL-6, and MIP-2 in TVX-treated mice as well as in mice treated with the fluoroquinolone levofloxacin, known to have a lower DILI-inducing profile. This supports the idea that early leukocyte recruitment regulates inflammation. In conclusion, disrupted regulation by leukocytes appears to constitute a fundamental step in the onset of TVX-induced liver injury, acting in concert with the capability of TVX to induce hepatocyte cell death. Interference of leukocyte-mediated regulation of inflammation represents a novel mechanism to explain the onset of DILI.

Tissue influx of neutrophils and monocytes is delayed during development of trovafloxacin-induced tumor necrosis factor-dependent liver injury in mice.

Idiosyncratic drug-induced liver injury (iDILI) has a poorly understood pathogenesis. However, iDILI is often associated with inflammatory stress signals in human patients as well as animal models. Tumor necrosis factor (TNF) and neutrophils play a key role in onset of trovafloxacin (TVX)-induced iDILI, but the exact role of neutrophils and other leukocytes remains to be defined. We therefore set out to study the kinetics of immunological changes during the development of TVX-induced iDILI in the established murine model of acute liver injury induced by administration of TVX and TNF. Initially, TNF stimulated the appearance of leukocytes, in particular neutrophils, into the liver of TVX-treated mice, but even more so in control mice treated with the non-DILI inducing analogue levofloxacin (LVX) or saline as vehicle (Veh). This difference was apparent at 2 hours after TNF administration, but at 4 hours, the relative neutrophil amounts were reduced again in Veh- and LVX-treated mice whereas the amounts in TVX-treated mice remained at the same increased level as at 2 hours. The influx of monocytes/macrophages, which was unaffected in Veh- and LVX-treated mice was markedly reduced or even absent in TVX-treated mice. Unlike controls, mice receiving TVX + TNF display severe hepatotoxicity with clear pathology and apoptosis, coagulated hepatic vessels and increased alanine aminotransferase levels and interleukin 6/10 ratios. Findings indicate that TVX delays the acute influx of neutrophils and monocytes/macrophages. Considering their known anti-inflammatory functions, the disruption of influx of these innate immune cells may hamper the resolution of initial cytotoxic effects of TVX and thus contribute to liver injury development.

A CK2-targeted Pt(IV) prodrug to disrupt DNA damage response.

A Pt(IV) prodrug, Cx-platin, containing CX-4945 (a CK2 inhibitor) as an axial ligand was designed and prepared by targeting CK2 to disrupt DNA damage response. In vitro study indicated that Cx-platin had superior cytotoxicity to cisplatin against a number of cancer cell lines with distinct CK2-expressed levels, caused CK2-overexpressed cancer cells death via suppressing CK2-mediated DNA damage repair and reversed cisplatin resistance. Mechanistic investigation suggested that the potent antitumor activity of Cx-platin resulted from its major suppression of CK2-phosphorylated MDC1 to combine FHA domain of aprataxin to DNA double strand breaks (DSBs) caused by improved cellular uptakes of Pt and ATM deactivation. Further in vivo tests exhibited that Cx-platin displayed high tumor inhibition rates, increased weight gain, and hardly toxicity effects in contrast to cisplatin.

Acyl-glucuronide as a Possible Cause of Trovafloxacin-Induced Liver Toxicity: Induction of Chemokine (C-X-C Motif) Ligand 2 by Trovafloxacin Acyl-glucuronide.

Trovafloxacin is an antibiotic that was withdrawn from the market relatively soon after its release due to the risk of hepatotoxicity. Trovafloxacin is mainly metabolized to its acyl-glucuronide by uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) 1A1. In this study, we examined whether the acyl-glucuronide is involved in the development of hepatotoxicity. A UGT1A1-induced cell model was developed and the toxicity of trovafloxacin acyl-glucuronide was evaluated. The UGT1A1-induced cell model was developed by treating HepG2 cells with chrysin for 48 h. Chemokine (C-X-C motif) ligand 2, a cytokine involved in drug-induced liver injury, was uniquely induced by trovafloxacin in the UGT1A1-induced HepG2 cells. Induction of UGT1A1 resulted in a decrease in cell viability. An in vivo animal study further demonstrated the importance of UGT1A1 in the trovafloxacin-induced liver toxicity. Although the complete mechanism of trovafloxacin-induced liver injury is still unknown, trovafloxacin acyl-glucuronide can be involved in the development of toxic reactions in vitro and in vivo.

Replacement of cardiotoxic aminopiperidine linker with piperazine moiety reduces cardiotoxicity? Mycobacterium tuberculosis novel bacterial topoisomerase inhibitors.

Recently numerous non-fluoroquinolone-based bacterial type II topoisomerase inhibitors from both the GyrA and GyrB classes have been reported as antibacterial agents. Inhibitors of the GyrA class include aminopiperidine-based novel bacterial type II topoisomerase inhibitors (NBTIs). However, inhibition of the cardiac ion channel remains a serious liability for the aminopiperidine based NBTIs. In this paper we replaced central aminopiperidine linker with piperazine moiety and tested for its biological activity. We developed a series of twenty four compounds with a piperazine linker 1-(2-(piperazin-1-yl)ethyl)-1,5-naphthyridin-2(1H)-one, by following a multistep protocol. Among them compound 4-(2-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)-N-(4-nitrophenyl)piperazine-1-carboxamide (11) was the most promising inhibitor with Mycobacterium tuberculosis (MTB) DNA gyrase enzyme supercoiling IC50 of 0.29±0.22µM, with a good MTB MIC of 3.45µM. These kind of compounds retains good potency and showed reduced cardiotoxicity compared to aminopiperidines.

Hepatotoxicants induce cytokine imbalance in response to innate immune system.

In recent years, attention has been paid to innate immune systems as mechanisms to initiate or promote drug-induced liver injury (DILI). Kupffer cells are hepatic resident macrophages and might be involved in the pathogenesis of DILI by release of pro- and anti-inflammatory mediators such as cytokines, chemokines, reactive oxygen species, and/or nitric oxides. The purpose of this study was to investigate alterations in mediator levels induced by hepatotoxic compounds in isolated Kupffer cells and discuss the relation between balance of each cytokine or chemokine and potential of innate immune-mediated DILI. Primary cultured rat Kupffer cells were treated with hepatotoxic (acetaminophen, troglitazone, trovafloxacin) or non-hepatotoxic (pioglitazone, levofloxacin) compounds with or without lipopolysaccharide (LPS). After 24 hr treatment, cell supernatants were collected and various levels of mediators released by Kupffer cells were examined. Although hepatotoxicants had no effect on the LPS-induced tumor necrosis factor-alpha (TNF-α) secretion, they enhanced the release of pro-inflammatory cytokine interleukin-1 beta (IL-1ß) and suppressed the anti-inflammatory cytokines interleukin-6 (IL-6) and interleukin-10 (IL-10) induced by LPS. These cytokine shifts were not associated with switching the phenotypes of M1 and M2 macrophages in Kupffer cells. In conclusion, the present study suggested that the levels of some specific cytokines are affected by DILI-related drugs with LPS stimulation, and imbalance between pro- and anti-inflammatory cytokines, induced by the up-regulation of IL-1ß and the down-regulation of IL-6 or IL-10, plays a key role in innate immune-mediated DILI.

Trovafloxacin-induced replication stress sensitizes HepG2 cells to tumor necrosis factor-alpha-induced cytotoxicity mediated by extracellular signal-regulated kinase and ataxia telangiectasia and Rad3-related.

Use of the fluoroquinolone antibiotic trovafloxacin (TVX) was restricted due to idiosyncratic, drug-induced liver injury (IDILI). Previous studies demonstrated that tumor necrosis factor-alpha (TNF) and TVX interact to cause death of hepatocytes in vitro that was associated with prolonged activation of c-Jun N-terminal kinase (JNK), activation of caspases 9 and 3, and DNA damage. The purpose of this study was to explore further the mechanism by which TVX interacts with TNF to cause cytotoxicity. Treatment with TVX caused cell cycle arrest, enhanced expression of p21 and impaired proliferation, but cell death only occurred after cotreatment with TVX and TNF. Cell death involved activation of extracellular signal-related kinase (ERK), which in turn activated caspase 3 and ataxia telangiectasia and Rad3-related (ATR), both of which contributed to cytotoxicity. Cotreatment of HepG2 cells with TVX and TNF caused double-strand breaks in DNA, and ERK contributed to this effect. Inhibition of caspase activity abolished the DNA strand breaks. The data suggest a complex interaction of TVX and TNF in which TVX causes replication stress, and the downstream effects are exacerbated by TNF, leading to hepatocellular death. These results raise the possibility that IDILI from TVX results from MAPK and ATR activation in hepatocytes initiated by interaction of cytokine signaling with drug-induced replication stress.

Trovafloxacin enhances lipopolysaccharide-stimulated production of tumor necrosis factor-α by macrophages: role of the DNA damage response.

Trovafloxacin (TVX) is a drug that has caused idiosyncratic, drug-induced liver injury (IDILI) in humans. In a murine model of IDILI, otherwise nontoxic doses of TVX and the inflammagen lipopolysaccharide (LPS) interacted to produce pronounced hepatocellular injury. The liver injury depended on a TVX-induced, small but significant prolongation of tumor necrosis factor-α (TNF) appearance in the plasma. The enhancement of TNF expression by TVX was reproduced in vitro in RAW 264.7 murine macrophages (RAW cells) stimulated with LPS. The current study was designed to identify the molecular target of TVX responsible for this response in RAW cells. An in silico analysis suggested a favorable binding profile of TVX to eukaryotic topoisomerase II-α (TopIIα), and a cell-free assay revealed that TVX inhibited eukaryotic TopIIα activity. Topoisomerase inhibition is known to lead to DNA damage, and TVX increased the DNA damage marker phosphorylated histone 2A.X in RAW cells. Moreover, TVX induced activation of the DNA damage sensor kinases, ataxia telangiectasia mutated (ATM) and Rad3-related (ATR). The ATR inhibitor NU6027 [6-(cyclohexylmethoxy)-5-nitrosopyrimidine-2,4-diamine] prevented the TVX-mediated increases in LPS-induced TNF mRNA and protein release, whereas a selective ATM inhibitor [2-(4-morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one (KU55933)] was without effect. TVX prolonged TNF mRNA stability, and this effect was largely attenuated by NU6027. These results suggest that TVX can inhibit eukaryotic topoisomerase, leading to activation of ATR and potentiation of TNF release by macrophages, at least in part through increased mRNA stability. This off-target effect might contribute to the ability of TVX to precipitate IDILI in humans.

Molecular mechanisms of hepatocellular apoptosis induced by trovafloxacin-tumor necrosis factor-alpha interaction.

Idiosyncratic drug-induced liver injury (IDILI) continues to be a significant human health problem. IDILI is characterized as occurring in a minority of individuals exposed to a drug, yet it accounts for as much as 17% of all cases of acute liver failure. Despite these concerns, the mechanisms underlying IDILI remain unknown. Trovafloxacin (TVX), which causes IDILI in humans, also causes hepatocellular death in vitro when combined with tumor necrosis factor-alpha (TNF) treatment. However, the molecular mechanisms involved in this toxicity are not fully characterized. The purpose of this study was to identify mechanisms by which TVX and TNF interact to cause hepatocellular death, with a focus on a human hepatocyte cell line. TVX and TNF interacted to cause cytotoxicity in HepG2 cells at drug concentrations similar to those in people undergoing TVX therapy. TVX/TNF treatment caused apoptosis and DNA damage in HepG2 cells that depended on caspase activation. Prolonged activation of JNK occurred in TVX/TNF-induced cytotoxicity, and treatment with the JNK selective inhibitor SP600125 attenuated cytotoxicity. TVX/TNF cotreatment also caused cytotoxicity in isolated primary murine hepatocytes that was dependent on caspase activation. These results increase understanding of molecular signaling pathways involved in hepatocellular death caused by a drug with idiosyncratic liability in the presence of TNF.

CK2 inhibitor CX4945 induces sequential inactivation of proteins in the signaling pathways related with cell migration and suppresses metastasis of A549 human lung cancer cells.

Casein kinase 2 (CK2) is known to be involved in various cellular processes such as cell cycle, apoptosis and proliferation. It has been reported that the inhibition of CK2 induced by recently developed small molecule CX4945 shows anti-cancer effects including anti-proliferation and anti-angiogenesis in several different cancers including prostate cancer. Here we report that migration and invasion of A549 human lung cancer cells are suppressed by the inhibition of CK2 induced by CX4945. We found that CX4945 sequentially attenuates the proteins in PI3K/Akt and MAPK pathways, two signaling pathways related with cell migration. This sequential control of signal pathways inhibits the expression of membrane type 1-matrix metalloproteinase and this leads to the selective attenuation of one of the gelatinases, MMP-2, which can degrade components of extracellular matrix, and metastasis of A549 human lung cancer cell.

Implication of hepatic transporters (MDR1 and MRP2) in inflammation-associated idiosyncratic drug-induced hepatotoxicity investigated by microvolume cytometry.

Idiosyncratic drug-induced hepatotoxicity accounts for about 13% of all cases of acute liver failure, therefore cited as the most frequent reason for post-marketing drug withdrawal. Despite this, the underlying mechanisms remain poorly understood due to lack in adequate screening assays and predictive in vitro models. Hepatic transporters play a crucial role in the absorption, distribution, and elimination of both endogenous substrates and xenobiotics. Defects in transporter function can lead to altered drug disposition, including toxicity and loss of efficacy. Inflammation is one condition for demonstrated variable drug response, attributed in part, to changes in function of drug transporters. The present study investigates the implication of two important hepatic transporters (MDR1 and MRP2) in idiosyncratic drug-induced hepatotoxicity in the presence and absence of an inflammatory context. The synergistic effect of idiosyncratic drugs (Trovafloxacin, nimesulide, telithromycin, and nefazodone) and inflammatory stimuli (TNF-α + LPS) on the efflux activity of hepatic transporters was studied using microvolume cytometry. Our results demonstrated on the one hand that both MDR1 and MRP2 are variably implicated in idiosyncratic drug-induced liver injury and on the other hand that the occurrence of an inflammatory reaction during idiosyncratic drug therapy can noticeably modulate this implication. In the absence of an inflammatory stress, none of the four tested drugs modulated the efflux activity of MRP2; nevertheless telithromycin and nefazodone inhibited the efflux activity of MDR1. Upon occurrence of an inflammatory stress, the inhibitory potential of trovafloxacin, nimesulide, and nefazodone on the efflux activity of MRP2 was noticeably revealed, while the telithromycin and nefazodone-induced inhibition of MDR1 was clearly attenuated. Knowledge of underlying mechanisms may significantly contribute to elimination of potential hepatotoxic drugs long before marketing and to prevention of drug-induced hepatotoxicity.

Novel dihydrobenzofuro[4,5-b][1,8]naphthyridin-6-one derivative, MHY-449, induces apoptosis and cell cycle arrest in HCT116 human colon cancer cells.

Colorectal cancer (CRC) is the second most frequent cancer in men and the third most common cancer in women in Korea. In spite of the significant advances in conventional therapeutic approaches to CRC, most patients ultimately die of their disease. There is a need to develop novel preventive approaches for this malignancy. This study was carried out to investigate the anticancer effect of the diastereoisomeric compounds, MHY-449 and MHY-450, novel dihydrobenzofuro[4,5-b][1,8]naphthyridin-6-one derivatives, on HCT116 human colon cancer cells. MHY-449 exhibited more potent cytotoxicity than MHY-450, against HCT116 cells. Treatment of cells with MHY-449 resulted in growth inhibition and induction of apoptosis in a concentration-dependent manner, and inhibition of proliferation in a time-dependent manner. The induction of apoptosis was observed by decreased cell viability, DNA fragmentation, activation of protein levels involved in death receptors. Moreover, activation of caspase-3, -8 and -9 and cleavage of poly(ADP-ribose) polymerase and alteration in the ratio of Bax/Bcl-2 protein expression was observed. MHY-449 induced G2/M phase arrest in the cell cycle progression which was observed by flow cytometry analysis, and a decrease in the protein expression of cyclin B1 and its activating partners Cdc25c and Cdc2. MHY-449 also caused increase in the expression levels of p53, a tumor suppressor gene, and p21WAF1/CIP and p27KIP, G2/M phase inhibitors. These results suggest that MHY-449 may be a useful candidate for chemo-prevention and/or treatment of colon cancer.