Phenotypic screen of sixty-eight colorectal cancer cell lines identifies CEACAM6 and CEACAM5 as markers of acid resistance.

Elevated cancer metabolism releases lactic acid and CO2 into the under-perfused tumor microenvironment, resulting in extracellular acidosis. The surviving cancer cells must adapt to this selection pressure; thus, targeting tumor acidosis is a rational therapeutic strategy to manage tumor growth. However, none of the major approved treatments are based explicitly on disrupting acid handling, signaling, or adaptations, possibly because the distinction between acid-sensitive and acid-resistant phenotypes is not clear. Here, we report pH-related phenotypes of sixty-eight colorectal cancer (CRC) cell lines by measuring i) extracellular acidification as a readout of acid production by fermentative metabolism and ii) growth of cell biomass over a range of extracellular pH (pHe) levels as a measure of the acid sensitivity of proliferation. Based on these measurements, CRC cell lines were grouped along two dimensions as "acid-sensitive"/"acid-resistant" versus "low metabolic acid production"/"high metabolic acid production." Strikingly, acid resistance was associated with the expression of CEACAM6 and CEACAM5 genes coding for two related cell-adhesion molecules, and among pH-regulating genes, of CA12. CEACAM5/6 protein levels were strongly induced by acidity, with a further induction under hypoxia in a subset of CRC lines. Lack of CEACAM6 (but not of CEACAM5) reduced cell growth and their ability to differentiate. Finally, CEACAM6 levels were strongly increased in human colorectal cancers from stage II and III patients, compared to matched samples from adjacent normal tissues. Thus, CEACAM6 is a marker of acid-resistant clones in colorectal cancer and a potential motif for targeting therapies to acidic regions within the tumors.

Acid-adapted cancer cells alkalinize their cytoplasm by degrading the acid-loading membrane transporter anion exchanger 2, SLC4A2.

Acidic environments reduce the intracellular pH (pHi) of most cells to levels that are sub-optimal for growth and cellular functions. Yet, cancers maintain an alkaline cytoplasm despite low extracellular pH (pHe). Raised pHi is thought to be beneficial for tumor progression and invasiveness. However, the transport mechanisms underpinning this adaptation have not been studied systematically. Here, we characterize the pHe-pHi relationship in 66 colorectal cancer cell lines and identify the acid-loading anion exchanger 2 (AE2, SLC4A2) as a regulator of resting pHi. Cells adapt to chronic extracellular acidosis by degrading AE2 protein, which raises pHi and reduces acid sensitivity of growth. Acidity inhibits mTOR signaling, which stimulates lysosomal function and AE2 degradation, a process reversed by bafilomycin A1. We identify AE2 degradation as a mechanism for maintaining a conducive pHi in tumors. As an adaptive mechanism, inhibiting lysosomal degradation of AE2 is a potential therapeutic target.

Solute exchange through gap junctions lessens the adverse effects of inactivating mutations in metabolite-handling genes.

Growth of cancer cells in vitro can be attenuated by genetically inactivating selected metabolic pathways. However, loss-of-function mutations in metabolic pathways are not negatively selected in human cancers, indicating that these genes are not essential in vivo. We hypothesize that spontaneous mutations in 'metabolic genes' will not necessarily produce functional defects because mutation-bearing cells may be rescued by metabolite exchange with neighboring wild-type cells via gap junctions. Using fluorescent substances to probe intercellular diffusion, we show that colorectal cancer (CRC) cells are coupled by gap junctions assembled from connexins, particularly Cx26. Cells with genetically inactivated components of pH regulation (SLC9A1), glycolysis (ALDOA), or mitochondrial respiration (NDUFS1) could be rescued through access to functional proteins in co-cultured wild-type cells. The effect of diffusive coupling was also observed in co-culture xenografts. Rescue was largely dependent on solute exchange via Cx26 channels, a uniformly and constitutively expressed isoform in CRCs. Due to diffusive coupling, the emergent phenotype is less heterogenous than its genotype, and thus an individual cell should not be considered as the unit under selection, at least for metabolite-handling processes. Our findings can explain why certain loss-of-function mutations in genes ascribed as 'essential' do not influence the growth of human cancers.

CRISPR-Cas9 screen identifies oxidative phosphorylation as essential for cancer cell survival at low extracellular pH.

Unlike most cell types, many cancer cells survive at low extracellular pH (pHe), a chemical signature of tumors. Genes that facilitate survival under acid stress are therefore potential targets for cancer therapies. We performed a genome-wide CRISPR-Cas9 cell viability screen at physiological and acidic conditions to systematically identify gene knockouts associated with pH-related fitness defects in colorectal cancer cells. Knockouts of genes involved in oxidative phosphorylation (NDUFS1) and iron-sulfur cluster biogenesis (IBA57, NFU1) grew well at physiological pHe, but underwent profound cell death under acidic conditions. We identified several small-molecule inhibitors of mitochondrial metabolism that can kill cancer cells at low pHe only. Xenografts established from NDUFS1-/- cells grew considerably slower than their wild-type controls, but growth could be stimulated with systemic bicarbonate therapy that lessens the tumoral acid stress. These findings raise the possibility of therapeutically targeting mitochondrial metabolism in combination with acid stress as a cancer treatment option.

Evidence-based guidelines for controlling pH in mammalian live-cell culture systems.

A fundamental variable in culture medium is its pH, which must be controlled by an appropriately formulated buffering regime, since biological processes are exquisitely sensitive to acid-base chemistry. Although awareness of the importance of pH is fostered early in the training of researchers, there are no consensus guidelines for best practice in managing pH in cell cultures, and reporting standards relating to pH are typically inadequate. Furthermore, many laboratories adopt bespoke approaches to controlling pH, some of which inadvertently produce artefacts that increase noise, compromise reproducibility or lead to the misinterpretation of data. Here, we use real-time measurements of medium pH and intracellular pH under live-cell culture conditions to describe the effects of various buffering regimes, including physiological CO2/HCO3- and non-volatile buffers (e.g. HEPES). We highlight those cases that result in poor control, non-intuitive outcomes and erroneous inferences. To improve data reproducibility, we propose guidelines for controlling pH in culture systems.

A high-throughput ratiometric method for imaging hypertrophic growth in cultured primary cardiac myocytes.

Maladaptive hypertrophy of cardiac myocytes increases the risk of heart failure. The underlying signaling can be triggered and interrogated in cultured neonatal ventricular myocytes (NRVMs) using sophisticated pharmacological and genetic techniques. However, the methods for quantifying cell growth are, by comparison, inadequate. The lack of quantitative, calibratable and computationally-inexpensive high-throughput technology has limited the scope for using cultured myocytes in large-scale analyses. We present a ratiometric method for quantifying the hypertrophic growth of cultured myocytes, compatible with high-throughput imaging platforms. Protein biomass was assayed from sulforhodamine B (SRB) fluorescence, and image analysis calculated the quotient of signal from extra-nuclear and nuclear regions. The former readout relates to hypertrophic growth, whereas the latter is a reference for correcting protein-independent (e.g. equipment-related) variables. This ratiometric measure, when normalized to the number of cells, provides a robust quantification of cellular hypertrophy. The method was tested by comparing the efficacy of various chemical agonists to evoke hypertrophy, and verified using independent assays (myocyte area, transcripts of markers). The method's high resolving power and wide dynamic range were confirmed by the ability to generate concentration-response curves, track the time-course of hypertrophic responses with fine temporal resolution, describe drug/agonist interactions, and screen for novel anti-hypertrophic agents. The method can be implemented as an end-point in protocols investigating hypertrophy, and is compatible with automated plate-reader platforms for generating high-throughput data, thereby reducing investigator-bias. Finally, the computationally-minimal workflow required for obtaining measurements makes the method simple to implement in most laboratories.

BRCA1 and BRCA2 tumor suppressors protect against endogenous acetaldehyde toxicity.

Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. Here, we investigate whether HR-compromised cells are sensitive to acetaldehyde, similarly to FANCD2-deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2, or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification, and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2-deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR-deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication-associated DNA damage, leading to checkpoint activation, G2/M arrest, and cell death. Acetaldehyde-arrested replication forks require BRCA2 and FANCD2 for protection against MRE11-dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2-deficient tumors and ex vivo in patient-derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP-ribose) polymerase (PARP) inhibitors. The work presented here therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2-deficient cells and tumors.

Medicinally Used Asarum Species: High-Resolution LC-MS Analysis of Aristolochic Acid Analogs and In vitro Toxicity Screening in HK-2 Cells.

Species of Asarum are used in traditional Chinese medicine and, similar to members of the genus Aristolochia, they contain aristolochic acid analogs (AAAs). These compounds are known for their nephrotoxic and carcinogenic effects. So far, the phytochemistry and nephrotoxicity of species of Asarum is not well studied. A high-resolution LC-MS-based metabolomic approach was used to study the phytochemical variation in medicinally used Asarum species. The cytotoxicity of the samples was assessed using human kidney (HK-2) cells. The majority of samples contained potentially nephrotoxic AAAs, including 9-methoxy aristolactam (AL) IV, AL I, and AL IV. These compounds were present in methanol as well as water extracts. AAAs were detected in all parts of the plant. The majority of the extracts were not cytotoxic to HK-2 cells at the doses tested. However, other mechanisms relating to aristolochic acid nephropathy and cancer development, such as DNA adduct formation may occur. The results of this study provide a model for assessing lesser-known plant species for toxicity.

FANCD2 limits replication stress and genome instability in cells lacking BRCA2.

The tumor suppressor BRCA2 plays a key role in genome integrity by promoting replication-fork stability and homologous recombination (HR) DNA repair. Here we report that human cancer cells lacking BRCA2 rely on the Fanconi anemia protein FANCD2 to limit replication-fork progression and genomic instability. Our results identify a new role of FANCD2 in limiting constitutive replication stress in BRCA2-deficient cells, thereby affecting cell survival and treatment responses.

Interplay between Fanconi anemia and homologous recombination pathways in genome integrity.

The Fanconi anemia (FA) pathway plays a central role in the repair of DNA interstrand crosslinks (ICLs) and regulates cellular responses to replication stress. Homologous recombination (HR), the error-free pathway for double-strand break (DSB) repair, is required during physiological cell cycle progression for the repair of replication-associated DNA damage and protection of stalled replication forks. Substantial crosstalk between the two pathways has recently been unravelled, in that key HR proteins such as the RAD51 recombinase and the tumour suppressors BRCA1 and BRCA2 also play important roles in ICL repair. Consistent with this, rare patient mutations in these HR genes cause FA pathologies and have been assigned FA complementation groups. Here, we focus on the clinical and mechanistic implications of the connection between these two cancer susceptibility syndromes and on how these two molecular pathways of DNA replication and repair interact functionally to prevent genomic instability.

LC-MS- and (1)H NMR-Based Metabolomic Analysis and in Vitro Toxicological Assessment of 43 Aristolochia Species.

Species of Aristolochia are used as herbal medicines worldwide. They cause aristolochic acid nephropathy (AAN), a devastating disease associated with kidney failure and renal cancer. Aristolochic acids I and II (1 and 2) are considered to be responsible for these nephrotoxic and carcinogenic effects. A wide range of other aristolochic acid analogues (AAAs) exist, and their implication in AAN may have been overlooked. An LC-MS- and (1)H NMR-based metabolomic analysis was carried out on 43 medicinally used Aristolochia species. The cytotoxicity and genotoxicity of 28 Aristolochia extracts were measured in human kidney (HK-2) cells. Compounds 1 and 2 were found to be the most common AAAs. However, AA IV (3), aristolactam I (4), and aristolactam BI (5) were also widespread. No correlation was found between the amounts of 1 or 2 and extract cytotoxicity against HK-2 cells. The genotoxicity and cytotoxicity of the extracts could be linked to their contents of 5, AA D (8), and AA IIIa (10). These results undermine the assumption that 1 and 2 are exclusively responsible for the toxicity of Aristolochia species. Other analogues are likely to contribute to their toxicity and need to be considered as nephrotoxic agents. These findings facilitate understanding of the nephrotoxic mechanisms of Aristolochia and have significance for the regulation of herbal medicines.

A multivariate approach linking reported side effects of clinical antidepressant and antipsychotic trials to in vitro binding affinities.

The vast majority of approved antidepressants and antipsychotics exhibit a complex pharmacology. The mechanistic understanding of how these psychotropic medications are related to adverse drug reactions (ADRs) is crucial for the development of novel drug candidates and patient adherence. This study aims to associate in vitro assessed binding affinity profiles (39 compounds, 24 molecular drug targets) and ADRs (n=22) reported in clinical trials of antidepressants and antipsychotics (n>59.000 patients) by the use of robust multivariate statistics. Orthogonal projection to latent structures (O-PLS) regression models with reasonable predictability were found for several frequent ADRs such as nausea, diarrhea, hypotension, dizziness, headache, insomnia, sedation, sleepiness, increased sweating, and weight gain. Results of the present study support many well-known pharmacological principles such as the association of hypotension and dizziness with α1-receptor or sedation with H1-receptor antagonism. Moreover, the analyses revealed novel or hardly investigated mechanisms for common ADRs including the potential involvement of 5-HT6-antagonism in weight gain, muscarinic receptor antagonism in dizziness, or 5-HT7-antagonism in sedation. To summarize, the presented study underlines the feasibility and value of a multivariate data mining approach in psychopharmacological development of antidepressants and antipsychotics.

Naturally occurring aristolochic acid analogues and their toxicities.

Covering: 1960 to 2013. Aristolochic acids are known for causing aristolochic acid nephropathy, a renal fibrosis often associated with urothelial carcinoma. Aristolochic acid I and II are considered to be the cause of these nephrotoxic and carcinogenic effects. However a variety of aristolochic acid analogues, including aristolactams and 4,5-dioxoaporphines have been reported. Their implications in aristolochic acid nephropathy have possibly been overlooked. In this report, in vivo and in vitro toxicity and mutagenicity of these three classes of compounds are discussed. Furthermore, the review gives an update of aristolochic acids, aristolactams and 4,5-dioxoaporphines reported between 2003 and 2013 and their biological activities.

Is aristolochic acid nephropathy a widespread problem in developing countries? A case study of Aristolochia indica L. in Bangladesh using an ethnobotanical-phytochemical approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Species of Aristolochia are associated with aristolochic acid nephropathy (AAN), a renal interstitial fibrosis and upper urinary tract cancer (UUC). Aristolochic acid nephropathy has been reported in ten countries but its true incidence is unknown and most likely underestimated. By combining an ethnobotanical and phytochemical approach we provide evidence for the risk of AAN occurring in Bangladesh. More specifically, we assess the intra-specific variation of aristolochic acid analogues in medicinally used Aristolochia indica samples from Bangladesh. MATERIALS AND METHODS: Ethnobotanical information was collected from 16 kavirajes (traditional healers) in different study locations in Bangladesh. Plant samples were obtained from native habitats, botanical gardens, herbal markets and pharmaceutical companies. The samples were extracted using 70% methanol and were analysed using LC-DAD-MS and (1)H-NMR. RESULTS: Roots as well as leaves are commonly used for symptoms such as snake bites and sexual problems. Among the informants knowledge about toxicity or side effects is very limited and Aristolochia indica is often administered in very high doses. Replacement of Aristolochia indica with other medicinal plants such as Rauvolfia serpentina (L.) Benth. ex Kurz was common. Aristolochia indica samples contained a variety of aristolochic acid analogues such as aristolochic acid I, aristolochic acid II, cepharadione A and related compounds. CONCLUSIONS: AAN cases are likely to occur in Bangladesh and more awareness needs to be raised about the health risks associated with the use of Aristolochia indica and other species of Aristolochia as herbal medicines.

Metabolomic analysis of Ranunculus spp. as potential agents involved in the etiology of equine grass sickness.

Identification of toxic or harmful agents continues to be a key goal in agricultural chemistry. This paper reports a metabolomic analysis of Ranunculus repens and related species, which were recently postulated to be cocausative agents in the etiology of equine grass sickness (EGS). Specifically, samples collected at EGS sites were compared with those from non-EGS sites. Furthermore, interspecific and seasonal variations and the species' response to edaphic and climatic factors were investigated. (1)H NMR spectroscopy in combination with multivariate data analysis was applied to the crude methanol extracts of the Ranunculus samples, as well as their chloroform fractions. Samples from EGS sites were significantly different from control samples. The metabolite composition varied greatly between different Ranunculus species. No significant changes could be observed between samples collected in different seasons. This work provides strong evidence that Ranunculus is involved in the etiology of EGS and has implications for agricultural management of pastures.