Hydrostatic pressure regulates CYP1A2 expression in human hepatocytes via a mechanosensitive aryl hydrocarbon receptor-dependent pathway.

Approximately 75% of xenobiotics are primarily eliminated through metabolism; thus the accurate scaling of metabolic clearance is vital to successful drug development. Yet, when data is scaled from in vitro to in vivo, hepatic metabolic clearance, the primary source of metabolism, is still commonly underpredicted. Over the past decades, with biophysics used as a key component to restore aspects of the in vivo environment, several new cell culture settings have been investigated to improve hepatocyte functionalities. Most of these studies have focused on shear stress, i.e., flow mediated by a pressure gradient. One potential conclusion of these studies is that hepatocytes are naturally "mechanosensitive," i.e., they respond to a change in their biophysical environment. We demonstrate that hepatocytes also respond to an increase in hydrostatic pressure that, we suggest, is directly linked to the lobule geometry and vessel density. Furthermore, we demonstrate that hydrostatic pressure improves albumin production and increases cytochrome P-450 (CYP) 1A2 expression levels in an aryl hydrocarbon-dependent manner in human hepatocytes. Increased albumin production and CYP function are commonly attributed to the impacts of shear stress in microfluidic experiments. Therefore, our results highlight evidence of a novel link between hydrostatic pressure and CYP metabolism and demonstrate that the spectrum of hepatocyte mechanosensitivity might be larger than previously thought.

Towards better models and mechanistic biomarkers for drug-induced gastrointestinal injury.

Adverse drug reactions affecting the gastrointestinal (GI) tract are a serious burden on patients, healthcare providers and the pharmaceutical industry. GI toxicity encompasses a range of pathologies in different parts of the GI tract. However, to date no specific mechanistic diagnostic/prognostic biomarkers or translatable pre-clinical models of GI toxicity exist. This review will cover the current knowledge of GI ADRs, existing biomarkers and models with potential application for toxicity screening/monitoring. We focus on the current gaps in our knowledge, the potential opportunities and recommend that a systematic approach is needed to identify mechanism-based GI biomarkers with potential for clinical translation.

An integrated characterization of serological, pathological, and functional events in doxorubicin-induced cardiotoxicity.

Many efficacious cancer treatments cause significant cardiac morbidity, yet biomarkers or functional indices of early damage, which would allow monitoring and intervention, are lacking. In this study, we have utilized a rat model of progressive doxorubicin (DOX)-induced cardiomyopathy, applying multiple approaches, including cardiac magnetic resonance imaging (MRI), to provide the most comprehensive characterization to date of the timecourse of serological, pathological, and functional events underlying this toxicity. Hannover Wistar rats were dosed with 1.25 mg/kg DOX weekly for 8 weeks followed by a 4 week off-dosing "recovery" period. Electron microscopy of the myocardium revealed subcellular degeneration and marked mitochondrial changes after a single dose. Histopathological analysis revealed progressive cardiomyocyte degeneration, hypertrophy/cytomegaly, and extensive vacuolation after two doses. Extensive replacement fibrosis (quantified by Sirius red staining) developed during the off-dosing period. Functional indices assessed by cardiac MRI (including left ventricular ejection fraction (LVEF), cardiac output, and E/A ratio) declined progressively, reaching statistical significance after two doses and culminating in "clinical" LV dysfunction by 12 weeks. Significant increases in peak myocardial contrast enhancement and serological cardiac troponin I (cTnI) emerged after eight doses, importantly preceding the LVEF decline to <50%. Troponin I levels positively correlated with delayed and peak gadolinium contrast enhancement, histopathological grading, and diastolic dysfunction. In summary, subcellular cardiomyocyte degeneration was the earliest marker, followed by progressive functional decline and histopathological manifestations. Myocardial contrast enhancement and elevations in cTnI occurred later. However, all indices predated "clinical" LV dysfunction and thus warrant further evaluation as predictive biomarkers.

Targeted inhibition of the FGF19-FGFR4 pathway in hepatocellular carcinoma; translational safety considerations.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death and new therapies are urgently required to treat this disease. Recent data suggest that the FGF19-FGFR4 axis may be a key driver in certain forms of HCC, making the pathway an interesting, emerging molecular target for potential therapeutic intervention. A complication is that, outside of malignant disease, FGFR4 plays an important physiological role in the regulation of hepatic bile acid (BA) synthesis. FGF19 signalling via FGFR4 suppresses de novo BA production in the liver, tightly maintaining hepatic and systemic levels of these detergent-like molecules at a physiological threshold and preventing pathological complications of raised BA levels, such as cholestatic liver injury and bile acid diarrhoea. In some cases of HCC, the malignant disease causes bile duct obstruction, preventing BA secretion from the liver and resulting in cholestasis. Here, the role of FGFR4 signalling in both HCC and BA homoeostasis is discussed. The potential effects of therapeutic FGF19-FGFR4 inhibition on human hepatobiliary/gastrointestinal physiology are considered along with the potential safety implications of FGF19-FGFR4 blockade in patients with HCC.

Reducing attrition in drug development: smart loading preclinical safety assessment.

Entry into the crucial preclinical good laboratory practice (GLP) stage of toxicology testing triggers significant R&D investment yet >20% of AstraZeneca's potential new medicines have been stopped for safety reasons in this GLP phase alone. How could we avoid at least some of these costly failures? An analysis of historical toxicities that caused stopping ('stopping toxicities') showed that >50% were attributable to target organ toxicities emerging within 2 weeks of repeat dosing or to acute cardiovascular risks. By frontloading 2-week repeat-dose toxicity studies and a comprehensive assessment of cardiovascular safety, we anticipate a potential 50% reduction in attrition in the GLP phase. This will reduce animal use overall, save significant R&D costs and improve drug pipeline quality.

Induction of heart valve lesions by small-molecule ALK5 inhibitors.

Aberrant signaling by transforming growth factor-ß (TGF-ß) and its type I (ALK5) receptor has been implicated in a number of human diseases and this pathway is considered a potential target for therapeutic intervention. Transforming growth factor-ß signaling via ALK5 plays a critical role during heart development, but the role of ALK5 in the adult heart is poorly understood. In the current study, the preclinical toxicology of ALK5 inhibitors from two different chemistry scaffolds was explored. Ten-week-old female Han Wistar rats received test compounds by the oral route for three to seven days. Both compounds induced histopathologic heart valve lesions characterized by hemorrhage, inflammation, degeneration, and proliferation of valvular interstitial cells. The pathology was observed in all animals, at all doses tested, and occurred in all four heart valves. Immunohistochemical analysis of ALK5 in rat hearts revealed expression in the valves, but not in the myocardium. Compared to control animals, protein levels of ALK5 were unchanged in the heart valves of treated animals. We also observed a physeal dysplasia in the femoro-tibial joint of rats treated with ALK5 inhibitors, a finding consistent with a pharmacological effect described previously with ALK5 inhibitors. Overall, these findings suggest that TGF-ß signaling via ALK5 plays a critical role in maintaining heart valve integrity.

Accumulation and distribution of doxorubicin in tumour spheroids: the influence of acidity and expression of P-glycoprotein.

PURPOSE: The intra-tumour distribution of anticancer drugs remains an important, but often under-estimated, influence on drug efficacy. Tumour acidity and the presence of efflux pumps were examined for their influence on the distribution of doxorubicin in a solid tumour model. METHODS: Anticancer drug distribution and overall accumulation was measured in tumour spheroids (TS) of varying sizes. The distribution profiles were examined in normoxic and hypoxic TS, the latter generating metabolic acidosis. Finally, the drug distribution profiles were related to efficacy using radial outgrowth assays. RESULTS: In large tumour spheroids (TS) (d ~500 µm), intracellular accumulation of doxorubicin was restricted to cells in the outermost layers and failed to accumulate within the viable cells in the 'intermediate' hypoxic zone. A similar profile was obtained for another protonatable amine, 7-AAD. In contrast, the distribution of the non-ionisable drug (at physiological pH) BODIPY-Taxol was uniform throughout the TS. In order to independently model the hypoxic and normoxic zones of TS, we compared drug accumulation in small entirely normoxic TS (d ~200 µm) with equivalent sized ones exposed to hypoxia in an anaerobic chamber. Exposure of TS to hypoxia caused a considerable reduction in the pH of the bathing medium and lower tissue accumulation of doxorubicin. Interstitial acidity reduces the proportion of doxorubicin in the non-ionised form. CONCLUSIONS: In TS, the accumulation and distribution of doxorubicin was influenced by both the expression of P-glycoprotein and hypoxia-induced acidity. Therefore, optimisation of doxorubicin chemotherapy for hypoxic tumours will require circumvention of both of these crucial pharmacokinetic determinants.

Cardiotoxicity associated with targeting kinase pathways in cancer.

Cardiotoxicity, also referred to as drug-induced cardiac injury, is an issue associated with the use of some small-molecule kinase inhibitors and antibody-based therapies targeting signaling pathways in cancer. Although these drugs have had a major impact on cancer patient survival, data have implicated kinase-targeting agents such as sunitinib, imatinib, trastuzumab, and sorafenib in adversely affecting cardiac function in a subset of treated individuals. In many cases, adverse cardiac events in the clinic were not anticipated based on preclinical safety evaluation of the molecule. In order to support the development of efficacious and safe kinase inhibitors for the treatment of cancer and other indications, new preclinical approaches and screens are required to predict clinical cardiotoxicity. Laboratory investigations into the underlying molecular mechanisms of heart toxicity induced by these molecules have identified potentially common themes including mitochondrial perturbation and modulation of adenosine monophosphate-activated protein kinase activity. Studies characterizing cardiac-specific kinase knockout mouse models have developed our understanding of the homeostatic role of some of these signaling mediators in the heart. Therefore, when considering kinases as potential future targets or when examining secondary pharmacological interactions of novel kinase inhibitors, these models may help to inform us of the potential adverse cardiac effects in the clinic.

Gender differences in drug toxicity.

Clinical data suggest that gender dimorphic profiles are emerging in terms of both drug efficacy and adverse drug reactions (ADRs). With an increasing emphasis on individualised therapies and the need to prevent drug attrition there is a compelling need to understand the molecular basis for gender dimorphic profiles in ADRs and the consequences. Classes of agents exhibiting gender-based variation in pharmaceutical efficacy and toxicity include anaesthetics, HIV-1 therapies and antiarrhythmic drugs. Body weight differences are often cited as a reason for differences in drug pharmacokinetics and subsequent toxicity. However, some studies accounted for these factors and still found significance suggesting that dosage versus body weight does not explain the outcome. Here, we present an overview of current understanding of gender-specific drug toxicity and present rational molecular explanations for these adverse events. There is mounting evidence in support of hormonal effects underpinning the majority of the ADR differences observed between the sexes.

Synchronised phosphorylation of BNIP3, Bcl-2 and Bcl-xL in response to microtubule-active drugs is JNK-independent and requires a mitotic kinase.

BNIP3 is a hypoxia-inducible BH3-only member of the Bcl-2 family of proteins that regulate apoptosis and autophagy. However the role of BNIP3 in the hypoxia response has proved difficult to define and remains controversial. In this study we show that in cancer cells, knockdown or forced expression of BNIP3 fails to modulate cell survival under hypoxic or normoxic conditions. However, we demonstrate that BNIP3 is regulated post-translationally, existing as multiple monomeric and dimeric phosphorylated forms. Upon treatment with microtubule inhibitors, but not other classes of chemotherapeutics, BNIP3 becomes hyperphosphorylated. We demonstrate that the phosphorylation of BNIP3 occurs in synchrony with phosphorylation of its binding partners Bcl-2 and Bcl-xL. Microtubule inhibitor-induced phosphorylation of these proteins occurs independently of the AKT/mTor and JNK kinase pathways and requires Mps1 mitotic checkpoint kinase activity. Inhibition of mitotic arrest in the presence of paclitaxel blocks the phosphorylation of BNIP3, Bcl-2 and Bcl-xL, demonstrating that these proteins are phosphorylated by a mitochondrially active mitotic kinase. We show that phosphorylation increases the stability of BNIP3 and that BNIP3 predominantly interacts with the phosphorylated form of Bcl-2. This study provides new insight into the post-translational functional control of these Bcl-2 family members.

The role of ATF4 stabilization and autophagy in resistance of breast cancer cells treated with Bortezomib.

The ubiquitin-proteasome system plays a key regulatory role in cellular homeostasis. The inhibition of the 26S proteasome by Bortezomib leads to the accumulation of misfolded proteins, resulting in endoplasmic reticulum stress followed by a coordinated cellular response called unfolded protein response (UPR). Endoplasmic reticulum stress is also a potent inducer of macroautophagy. Bortezomib is a selective and potent inhibitor of the 26S proteasome and is approved for the treatment of multiple myeloma. Clinical trials with Bortezomib have shown promising results for some types of cancers, but not for some others, including those of the breast. In this study, we show that Bortezomib induces the UPR and autophagy in MCF7 breast cancer cells. Surprisingly, Bortezomib did not induce phosphorylation of PERK, a key initial step of the UPR. We show that induction of autophagy by Bortezomib is dependent on the proteasomal stabilisation of ATF4 and up-regulation of LC3B by ATF4. We show that ATF4 and LC3B play a critical role in activating autophagy and protecting cells from Bortezomib-induced cell death. Our experiments also reveal that HDAC6 knockdown results in decreased LC3B protein and reduced autophagy. Our work shows that the induction of autophagy through ATF4 may be an important resistance mechanism to Bortezomib treatment in breast cancer, and targeting autophagy may represent a novel approach to sensitize breast cancers to Bortezomib.

Resistance to chemotherapy in cancer: a complex and integrated cellular response.

Inherent and acquired resistance pathways account for the high rate of failure in cancer chemotherapy. The mechanisms or pathways mediating resistance may be classified as pharmacokinetic (i.e. alter intratumour drug exposue) or pharmacodynamic (i.e. failure to elicit cytotoxicity). More often than not, the resistant phenotype is characterised by alterations in multiple pathways. Consequently, the pathways may act synergistically or generate a broad spectrum of resistance to anticancer drugs. There has been a great deal of systematic characterisation of drug resistance in vitro. However, translating this greater understanding into clinical efficacy has rarely been achieved. This review explores the phenomenon of drug resistance in cancer and highlights the gap between in vitro and in vivo observations. This gap presents a major obstacle in overcoming drug resistance and restoring sensitivity to chemotherapy.

The role of the hypoxia-inducible BH3-only proteins BNIP3 and BNIP3L in cancer.

In this review, we summarize current knowledge of the biological functions of the atypical BH3-only proteins BNIP3 and BNIP3L, focusing on the role of these proteins in cancer. Hypoxia increases the expression of BNIP3 through the transcription factor HIF-1, but despite a considerable number of investigations, it has proven difficult to establish a clear role for BNIP3 in the cellular hypoxic response. BNIP3 can induce a form of cell death that shows features of both necrosis and apoptosis, but unusually for a BH3-only protein, death occurs independently of the BH3 domain and is critically dependent on a C-terminal transmembrane domain, which also localizes the protein to the mitochondria. BNIP3 expression does not always result in cell death, suggesting that additional factors may suppress BNIP3 or cooperate with it to induce death. BNIP3 is highly expressed in some tumors, including those of the breast, lung and cervix. However, in colorectal and pancreatic cancers BNIP3 is frequently epigenetically silenced, possibly reflecting different functions for BNIP3 in different tissues. Recent reports have shown that BNIP3 can induce autophagy and there is some evidence to suggest this may represent an emerging role for BH3-only proteins in general. However, the mechanism through which BNIP3 induces autophagy and the cellular consequences of this are yet to be established.

Diffusivity and distribution of vinblastine in three-dimensional tumour tissue: experimental and mathematical modelling.

The distribution of chemotherapeutics in solid tumours is poorly understood and the contribution it makes to treatment failure is unknown. Novel approaches are required to understand how the three-dimensional organisation of cancer cells in solid tumours affects drug availability. Since convective drug transport is limited by increased interstitial pressure in poorly vascularised cancers, the aim of this study was to measure the diffusive hindrance exerted by solid tumour tissue. Multicell layer tumour models comprising DLD1 colon cancer cells were characterised and fluxes were determined for [3H]-vinblastine and [14C]-sucrose. The mathematical models provided the diffusion coefficients for both compounds and predicted higher exposure of cells in the vicinity of vessels. The diffusion of vinblastine was three times slower than that of sucrose. Although slow diffusion delays vinblastine penetration into the avascular regions of tumours, the proliferating cells are generally in the marginal area of tumours. The mathematical model that we have developed enabled accurate quantification of drug pharmacokinetic behaviour, in particular, the diffusivity of vinblastine within solid tissue. This mathematical model may be adapted readily to incorporate the influence of factors mediating pharmacokinetic drug resistance.

Modulation of multidrug resistance efflux pump activity to overcome chemoresistance in cancer.

Early publications using cultured cancer cells immediately recognized the phenomenon of resistance to anticancer agents. However, it was not until 1973 that it was first demonstrated that a major factor in the resistance of cancer cells was that of reduced drug accumulation. This year marks the 30th anniversary of the discovery by Juliano and Ling that P-glycoprotein mediates this active efflux of chemotherapeutic drugs from cancer cells. Since this seminal finding, the investigation of P-glycoprotein (MDR1, ATP binding cassette [ABC]B1) has proceeded with great vigour. However, it soon became apparent that P-glycoprotein was not expressed in all drug-resistant cells that displayed an accumulation deficiency, which led to the discovery of other ABC transporters involved in drug efflux. In 1992, the multidrug resistance-associated protein (MRP1, ABCC1) was identified in small cell lung cancer followed by breast cancer resistance protein (mitoxantrone resistance protein, ABCG2) in 1999. After three decades of research, can we confidently define the contribution of multidrug resistance transporters to chemoresistance and do we have clinically useful drugs to sensitise cancers?

Cellular effects of deoxynojirimycin analogues: uptake, retention and inhibition of glycosphingolipid biosynthesis.

Deoxynojirimycin (DNJ) analogues are inhibitors of ceramide glucosyltransferase (CGT), which catalyses the first step in the glucosphingolipid (GSL) biosynthetic pathway. We have synthesized a series of DNJ analogues to study the contribution of N-alk(en)yl side chains (C4, C9 or C18) to the behaviour of these analogues in cultured HL60 cells. When cells were treated for 16 h at non-cytotoxic concentrations of inhibitor, a 40-50% decrease in GSL levels was measured by HPLC analysis of GSL-derived oligosaccharides following ceramide glycanase digestion of GSL and 2-aminobenzamide labelling of the released oligosaccharides. Using a novel technique for short-term [14C]galactose labelling of cellular GSL, we used compound inhibition of GSL biosynthesis as a marker for compound uptake into cells. Surprisingly, the uptake of all three of the DNJ analogues was extremely rapid and was not dependent upon the length of the N-alk(en)yl moiety. Compound uptake occurred in less than 1 min, as shown by the complete inhibition of GSL labelling in cells treated with all the DNJ analogues. Greatly increased cellular retention of N-cis-13-octadecenyl-DNJ was observed relative to the shorter-chain compounds, N-butyl-DNJ and N-nonyl-DNJ, as indicated by complete inhibition of CGT 24 h after removal of inhibitor from the culture medium. The present study further characterizes the properties of N-alk(en)ylated DNJs, and demonstrates that increasing the length of the side chain is a simple way of improving imino sugar retention and therefore inhibitory efficacy for CGT in cultured cells.

Cellular effects of deoxynojirimycin analogues: inhibition of N-linked oligosaccharide processing and generation of free glucosylated oligosaccharides.

In the accompanying paper [Mellor, Neville, Harvey, Platt, Dwek and Butters (2004) Biochem. J. 381, 861-866] we treated HL60 cells with N-alk(en)yl-deoxynojirimycin (DNJ) compounds to inhibit glucosphingolipid (GSL) biosynthesis and identified a number of non-GSL-derived, small, free oligosaccharides (FOS) most likely produced due to inhibition of the oligosaccharide-processing enzymes a-glucosidases I and II. When HL60 cells were treated with concentrations of N-alk(en)ylated DNJ analogues that inhibited GSL biosynthesis completely, N-butyl- and N-nonyl-DNJ inhibited endoplasmic reticulum (ER) glucosidases I and II, but octadecyl-DNJ did not, probably due to the lack of ER lumen access for this novel, long-chain derivative. Glucosidase inhibition resulted in the appearance of free Glc1-3Man structures, which is evidence of Golgi glycoprotein endomannosidase processing of oligosaccharides with retained glucose residues. Additional large FOS was also detected in cells following a 16 h treatment with N-butyl- and N-nonyl-DNJ. When these FOS structures (>30, including >20 species not present in control cells) were characterized by enzyme digests and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS, all were found to be polymannose-type oligosaccharides, of which the majority were glucosylated and had only one reducing terminal GlcNAc (N-acetylglucosamine) residue (FOS-GlcNAc1), demonstrating a cytosolic location. These results support the proposal that the increase in glucosylated FOS results from enzyme-mediated cytosolic cleavage of oligosaccharides from glycoproteins exported from the ER because of misfolding or excessive retention. Importantly, the present study characterizes the cellular properties of DNJs further and demonstrates that side-chain modifications allow selective inhibition of protein and lipid glycosylation pathways. This represents the most detailed characterization of the FOS structures arising from ER a-glucosidase inhibition to date.

Small-molecule therapeutics for the treatment of glycolipid lysosomal storage disorders.

Glycosphingolipid (GSL) lysosomal storage disorders are a small but challenging group of human diseases to treat. Although these disorders appear to be monogenic in origin, where the catalytic activity of enzymes in GSL catabolism is impaired, the clinical presentation and severity of disease are heterogeneous. Present attitudes to treatment demand individual therapeutics designed to match the specific disease-related gene defect; this is an acceptable approach for those diseases with high frequency, but it lacks viability for extremely rare conditions. An alternative therapeutic approach termed 'substrate deprivation' or 'substrate reduction therapy' (SRT) aims to balance cellular GSL biosynthesis with the impairment in catalytic activity seen in lysosomal storage disorders. The development of N-alkylated iminosugars that have inhibitory activity against the first enzyme in the pathway for glucosylating sphingolipid in eukaryotic cells, ceramide-specific glucosyltransferase, offers a generic therapeutic for the treatment of all glucosphingolipidoses. The successful use of N-alkylated iminosugars to establish SRT as an alternative therapeutic strategy has been demonstrated in in vitro, in vivo and in clinical trials for type 1 Gaucher disease. The implications of these studies and the prospects of improvement to the design of iminosugar compounds for treating Gaucher and other GSL lysosomal storage disorders will be discussed.