Role of the cystathionine ß-synthase / H2S pathway in the development of cellular metabolic dysfunction and pseudohypoxia in down syndrome.

BACKGROUND: Overexpression of the transsulfuration enzyme cystathionine-ß-synthase (CBS), and overproduction of its product, hydrogen sulfide (H2S) are recognized as potential pathogenetic factors in Down syndrome (DS). The purpose of the study was to determine how the mitochondrial function and core metabolic pathways are affected by DS and how pharmacological inhibition of CBS affects these parameters. METHODS: 8 human control and 8 human DS fibroblast cell lines have been subjected to bioenergetic and fluxomic and proteomic analysis with and without treatment with a pharmacological inhibitor of CBS. RESULTS: DS cells exhibited a significantly higher CBS expression than control cells, and produced more H2S. They also exhibited suppressed mitochondrial electron transport and oxygen consumption and suppressed Complex IV activity, impaired cell proliferation and increased ROS generation. Inhibition of H2S biosynthesis with aminooxyacetic acid reduced cellular H2S, improved cellular bioenergetics, attenuated ROS and improved proliferation. 13C glucose fluxomic analysis revealed that DS cells exhibit a suppression of the Krebs cycle activity with a compensatory increase in glycolysis. CBS inhibition restored the flux from glycolysis to the Krebs cycle and reactivated oxidative phosphorylation. Proteomic analysis revealed no CBS-dependent alterations in the expression level of the enzymes involved in glycolysis, oxidative phosphorylation and the pentose phosphate pathway. DS was associated with the dysregulation of several components of the autophagy network; CBS inhibition normalized several of these parameters. CONCLUSIONS: Increased H2S generation in DS promotes pseudohypoxia and contributes to cellular metabolic dysfunction by causing a shift from oxidative phosphorylation to glycolysis.

Physiological concentrations of cyanide stimulate mitochondrial Complex IV and enhance cellular bioenergetics.

In mammalian cells, cyanide is viewed as a cytotoxic agent, which exerts its effects through inhibition of mitochondrial Complex IV (Cytochrome C oxidase [CCOx]). However, the current report demonstrates that cyanide's effect on CCOx is biphasic; low (nanomolar to low-micromolar) concentrations stimulate CCOx activity, while higher (high-micromolar) concentrations produce the "classic" inhibitory effect. Low concentrations of cyanide stimulated mitochondrial electron transport and elevated intracellular adenosine triphosphate (ATP), resulting in the stimulation of cell proliferation. The stimulatory effect of cyanide on CCOx was associated with the removal of the constitutive, inhibitory glutathionylation on its catalytic 30- and 57-kDa subunits. Transfer of diluted Pseudomonas aeruginosa (a cyanide-producing bacterium) supernatants to mammalian cells stimulated cellular bioenergetics, while concentrated supernatants were inhibitory. These effects were absent with supernatants from mutant Pseudomonas lacking its cyanide-producing enzyme. These results raise the possibility that cyanide at low, endogenous levels serves regulatory purposes in mammals. Indeed, the expression of six putative mammalian cyanide-producing and/or -metabolizing enzymes was confirmed in HepG2 cells; one of them (myeloperoxidase) showed a biphasic regulation after cyanide exposure. Cyanide shares features with "classical" mammalian gasotransmitters NO, CO, and H2S and may be considered the fourth mammalian gasotransmitter.

Meta-analysis of metabolites involved in bioenergetic pathways reveals a pseudohypoxic state in Down syndrome.

Clinical observations and preclinical studies both suggest that Down syndrome (DS) may be associated with significant metabolic and bioenergetic alterations. However, the relevant scientific literature has not yet been systematically reviewed. The aim of the current study was to conduct a meta-analysis of metabolites involved in bioenergetics pathways in DS to conclusively determine the difference between DS and control subjects. We discuss these findings and their potential relevance in the context of pathogenesis and experimental therapy of DS. Articles published before July 1, 2020, were identified by using the search terms "Down syndrome" and "metabolite name" or "trisomy 21" and "metabolite name". Moreover, DS-related metabolomics studies and bioenergetics literature were also reviewed. 41 published reports and associated databases were identified, from which the descriptive information and the relevant metabolomic parameters were extracted and analyzed. Mixed effect model revealed the following changes in DS: significantly decreased ATP, CoQ10, homocysteine, serine, arginine and tyrosine; slightly decreased ADP; significantly increased uric acid, succinate, lactate and cysteine; slightly increased phosphate, pyruvate and citrate. However, the concentrations of AMP, 2,3-diphosphoglycerate, glucose, and glutamine were comparable in the DS vs. control populations. We conclude that cells of subjects with DS are in a pseudo-hypoxic state: the cellular metabolic and bio-energetic mechanisms exhibit pathophysiological alterations that resemble the cellular responses associated with hypoxia, even though the supply of the cells with oxygen is not disrupted. This fundamental alteration may be, at least in part, responsible for a variety of functional deficits associated with DS, including reduced exercise difference, impaired neurocognitive status and neurodegeneration.

The phytohormone forchlorfenuron decreases viability and proliferation of malignant mesothelioma cells in vitro and in vivo.

Malignant mesothelioma (MM) is one of the most aggressive cancer types with a patient's life expectancy of typically less than one year upon diagnosis. The urgency of finding novel therapeutic approaches to treat mesothelioma is evident. Here we tested the effect of the plant-growth regulator forchlorfenuron (FCF), an inhibitor of septin function(s) in mammalian cells, on the viability and proliferation of MM cell lines, as well as other tumor cell lines derived from lung, prostate, colon, ovary, cervix and breast. Exposure to FCF strongly inhibited proliferation of human and mouse (most efficiently epithelioid) MM cells and all other tumor cells in a concentration-dependent manner and led to cell cycle arrest and cell death. The role of septin 7 (SEPT7), a presumably essential target of FCF in MM cells was confirmed by an shRNA strategy. FCF was robustly inhibiting tumor cell growth in vitro at low micromolar (IC50: ≈20-60µM) concentrations and more promisingly also in vivo. Initial experiments with FCF analogous revealed the importance of FCF's chloride group for efficient cell growth inhibition. FCF's rather low systemic toxicity might warrant for an extended search for other related and possibly more potent FCF analogues to target MM and putatively other septin-dependent tumors.

Using data on snus use in Sweden to compare different modelling approaches to estimate the population health impact of introducing a smoke-free tobacco product.

BACKGROUND: We have developed an approach for modelling the health impact of introducing new smoke-free tobacco products. We wished to compare its estimates with those of alternative approaches, when applied to snus, used in Sweden for many years. METHODS: Modelling was restricted to men aged 30-79 years for 1980-2009 and to four smoking-related diseases. Mortality data were extracted for Sweden and other European countries. Published data provided Swedish prevalence estimates for combinations of never/former/current smoking and snus use, and smoking prevalence estimates for other European countries. Approach 1 compares mortality in Sweden and in other countries with a smoking prevalence similar to Sweden's prevalence of combined smoking/snus use. Approaches 2 and 3 compare mortality in Sweden with hypothetical mortality had snus users smoked. Approach 3 uses our health impact model, individuals starting with the tobacco prevalence of Sweden in 1980. Tobacco histories during 30-year follow-up were then estimated using transition probabilities, with risk derived using a negative exponential model. Approach 2 uses annual tobacco prevalence estimates coupled with estimates of relative risk of current and former smokers regardless of history. The main applications of Approaches 2 and 3 assume that only smoking affects mortality, though sensitivity analyses using Approach 3 allow for risk to vary in snus users and dual users. RESULTS: Using Approach 2, estimated mortality increases in Sweden in 1980-2009 had snus not been introduced were: lung cancer 8786; COPD 1781; IHD 10,409; stroke 1720. The main Approach 3 estimates were similar (7931, 1969; 12,501; 1901). They decreased as risk in snus users and dual users increased. Approach 1 estimates differed wildly (77,762, 32,538; 77,438; 76,946), remaining very different following correction for differences between Sweden and the comparison countries in non-smoking-related disease mortality. CONCLUSIONS: Approach 1 is unreliable, accounting inadequately for non-tobacco factors affecting mortality. Approaches 2 and 3 provide reasonably similar approximate estimates of the mortality increase had snus not been available, but have differing advantages and disadvantages. Only Approach 3 considers tobacco history, but develops histories using tobacco transition probabilities, which is possibly less reliable than using estimated tobacco prevalences at each follow-up year.

Albumin evokes Ca2+-induced cell oxidative stress and apoptosis through TRPM2 channel in renal collecting duct cells reduced by curcumin.

In proteinuric nephropathies of chronic kidney disease, the epithelial cells of the nephron including the collecting duct are exposed to high concentrations of luminal albumin. Albumin is taken up from collecting duct cells by endocytosis causing excessive reactive oxygen species (ROS) production and a proinflammatory response. Curcumin used in the traditional medicine possesses anti-inflammatory and antioxidant effects. ROS and ADP-ribose (ADPR) activate the cation channel TRPM2. We hypothesize, that albumin-induced cell stress and proinflammatory response are mediated by Ca2+ and can be reduced by curcumin. The cortical collecting duct (CCD) cells mpkCCDc14 exhibit spontaneous and inducible Ca2+ oscillations, which can be blocked by pre-treatment with curcumin. Curcumin accumulates in plasma membrane and intracellular vesicles, where it interferes with TRPM2 and decreases the influx of Ca2+. Albumin reduces cell viability and increases apoptosis, NF-κB activation, and mitochondrial membrane depolarization via Ca2+-dependent signaling, which results in increased ROS production. Albumin-induced cell stress is diminished by the inhibition of TRPM2 after administration of curcumin and ADPR (PARP1) inhibitors. Curcumin did not reduce the Ca2+ elevation induced by thapsigargin in Ca2+-free medium, but it reduced the function of store-operated Ca2+ channels and ATP-evoked Ca2+ response. In conclusion, albumin-induced oxidative stress is mediated by Ca2+-dependent signaling via TRPM2 and leads to cell damage and a proinflammatory response, strengthening the role of CCD cells in the progression of chronic kidney disease.

ADP-Ribose and oxidative stress activate TRPM8 channel in prostate cancer and kidney cells.

Activation of TRPM8 channel through oxidative stress may induce Ca2+ and pro-apoptotic signals in prostate cancer and kidney cells. The aim of this study was to evaluate activation of TRPM8 can increase apoptosis and oxidative stress in the prostate cancer (Du145M8), TRPM8 knock out (Du 145M8KO), transfected (HEK293TM8) and non-transfected human kidney (HEK293) cells. Intracellular Ca2+ responses to TRPM8 activation were increased in the Du145M8 and HEK293TM8 cells from coming cumene hydrogen peroxide (CHPx), menthol, ADP-Ribose (ADPR), but not in the HEK293 and Du 145M8KO cells. The intracellular Ca2+ responses to both ADPR and CHPx were totally inhibited by the thiol cycle antioxidant glutathione, and TRPM8 blockers (N-(p-amylcinnamoyl)anthranilic acid and capsazepine). Apoptosis, Annexin V, mitochondrial membrane depolarization, intracellular ROS, caspase 3 and 9 values were increased through TRPM8 activation in the Du 145M8 but not in the Du 145M8KO and non-transfected HEK293 cells by CHPx and hydrogen peroxide. In conclusion, apoptotic and oxidant effects on the cells were increased activation of TRPM8 by oxidative stress and ADPR. Activation of TRPM8 through oxidative stress and ADPR in the cells could be used as an effective strategy in the treatment of prostate cancer cells.

Calretinin promotes invasiveness and EMT in malignant mesothelioma cells involving the activation of the FAK signaling pathway.

Calretinin (CR) is used as a positive marker for human malignant mesothelioma (MM) and is essential for mesothelioma cell growth/survival. Yet, the putative role(s) of CR during MM formation in vivo, binding partners or CR's influence on specific signaling pathways remain unknown. We assessed the effect of CR overexpression in the human MM cell lines MSTO-211H and SPC111. CR overexpression augmented the migration and invasion of MM cells in vitro. These effects involved the activation of the focal adhesion kinase (FAK) signaling pathway, since levels of total FAK and phospho-FAK (Tyr397) were found up-regulated in these cells. CR was also implicated in controlling epithelial-to-mesenchymal transition (EMT), evidenced by changes of the cell morphology and up-regulation of typical EMT markers. Co-IP experiments revealed FAK as a new binding partner of CR. CR co-localized with FAK at focal adhesion sites; moreover, CR-overexpressing cells displayed enhanced nuclear FAK accumulation and an increased resistance towards the FAK inhibitor VS-6063. Finally, CR downregulation via a lentiviral shRNA against CR (CALB2) resulted in a significantly reduced tumor formation in vivo in an orthotopic xenograft mouse model based on peritoneal MM cell injection. Our results indicate that CR might be considered as a possible target for MM treatment.

Absence of calretinin protein expression in malignant mesotheliomas from asbestos-exposed NF2+/- mice and mouse mesothelioma cell lines from various mouse strains.

BACKGROUND: Calretinin is the most widespread positive marker for the immunohistochemical identification of malignant mesothelioma (MM) and was proposed to serve as a blood-based biomarker. Functionally, evidence has accumulated that calretinin might be implicated in MM tumorigenesis. We aimed to identify calretinin (CR; Calb2) in murine MM and reactive mesothelial cells in granuloma from asbestos-exposed NF2+/- mice, a line heterozygous for the tumor suppressor merlin (NF2), used as a mouse MM model. Additionally, we sought to ascertain the presence of calretinin in MM cell lines from other mouse strains. We also intended to investigate the role of calretinin in mesotheliomagenesis by comparing the survival of asbestos-exposed NF2+/- and NF2+/-CR-/- mice. METHODS: NF2+/- and NF2+/-CR-/- mice, both lines on a C57Bl/6J background, were exposed to asbestos following an established protocol. Tumor histology and asbestos-induced mortality were assessed. MM and granuloma from NF2+/- mice were analyzed with immunohistochemical methods for calretinin expression. Levels of Calb2 mRNA and calretinin expression in tumors and MM cell lines of various mouse strains were determined by RT-qPCR and Western blot analysis, respectively. RESULTS: No expression of calretinin at the protein level was detected, neither in MM from NF2+/- mice, NF2+/- MM-derived cell lines nor immortalized mesothelial cells of mouse origin. At the mRNA level we detected Calb2 expression in MM cell lines from different mouse strains. Survival of NF2+/- and NF2+/-CR-/- mice exposed to asbestos showed no significant difference in a log-rank (Kaplan-Meier) comparison. CONCLUSIONS: The concomitant determination of calretinin and mesothelin blood levels has been proposed for early detection of human MM. Mouse MM models based on asbestos exposure are assumed to yield helpful information on the time course of appearance of mesothelin and calretinin in the blood of asbestos-treated mice determining the earliest time point for interventions. However, the observed absence of calretinin in MM from NF2+/- mice and derived cell lines, as well as from MM cells from Balb/c and C3H mice likely precludes the use of calretinin as a biomarker for mouse MM. The results also indicate possible species differences with respect to an involvement of calretinin in the formation of MM.

Putative cancer stem cells may be the key target to inhibit cancer cell repopulation between the intervals of chemoradiation in murine mesothelioma.

BACKGROUND: Cancer cell repopulation during chemotherapy or radiotherapy is a major factor limiting the efficacy of treatment. Cancer stem cells (CSC) may play critical roles during this process. We aim to demonstrate the role of mesothelioma stem cells (MSC) in treatment failure and eventually to design specific target therapies against MSC to improve the efficacy of treatment in malignant mesothelioma. METHODS: Murine mesothelioma AB12 and RN5 cells were used to compare tumorigenicity in mice. The expression of CSC-associated genes was evaluated by quantitative real-time PCR in both cell lines treated with chemo-radiation. Stemness properties of MSC-enriched RN5-EOS-Puro2 cells were characterized with flow cytometry and immunostaining. A MSC-specific gene profile was screened by microarray assay and confirmed thereafter. Gene Ontology analysis of the selected genes was performed by GOMiner. RESULTS: Tumor growth delay of murine mesothelioma AB12 cells was achieved after each cycle of cisplatin treatment, however, tumors grew back rapidly due to cancer cell repopulation between courses of chemotherapy. Strikingly, a 10-times lower number of irradiated cells in both cell lines led to a similar tumor incidence and growth rate as with untreated cells. The expression of CSC-associated genes such as CD24, CD133, CD90 and uPAR was dramatically up-regulated, while others did not change significantly after chemoradiation. Highly enriched MSC after selection with puromycin displayed an increasing GFP-positive population and showed typical properties of stemness. Comparatively, the proportion of MSC significantly increased after RN5-EOS parental cells were treated with either chemotherapy, γ-ray radiation, or a combination of the two, while MSC showed more resistance to the above treatments. A group of identified genes are most likely MSC-specific, and major pathways related to regulation of cell growth or apoptosis are involved. Upregulation of the gene transcripts Tnfsf18, Serpinb9b, Ly6a, and Nppb were confirmed. CONCLUSION: Putative MSC possess the property of stemness showing more resistance to chemoradiation, suggesting that MSC may play critical roles in cancer cell repopulation. Further identification of selected genes may be used to design novel target therapies against MSC, so as to eliminate cancer cell repopulation in mesothelioma.

Regulation of calretinin in malignant mesothelioma is mediated by septin 7 binding to the CALB2 promoter.

BACKGROUND: The calcium-binding protein calretinin (gene name: CALB2) is currently considered as the most sensitive and specific marker for the diagnosis of malignant mesothelioma (MM). MM is a very aggressive tumor strongly linked to asbestos exposure and with no existing cure so far. The mechanisms of calretinin regulation, as well as its distinct function in MM are still poorly understood. METHODS: We searched for transcription factors binding to the CALB2 promoter and modulating calretinin expression. For this, DNA-binding assays followed by peptide shotgun-mass spectroscopy analyses were used. CALB2 promoter activity was assessed by dual-luciferase reporter assays. Furthermore, we analyzed the effects of CALB2 promoter-binding proteins by lentiviral-mediated overexpression or down-regulation of identified proteins in MM cells. The modulation of expression of such proteins by butyrate was determined by subsequent Western blot analysis. Immunohistochemical analysis of embryonic mouse lung tissue served to verify the simultaneous co-expression of calretinin and proteins interacting with the CALB2 promoter during early development. Finally, direct interactions of calretinin with target proteins were evidenced by co-immunoprecipitation experiments. RESULTS: Septin 7 was identified as a butyrate-dependent transcription factor binding to a CALB2 promoter region containing butyrate-responsive elements (BRE) resulting in decreased calretinin expression. Accordingly, septin 7 overexpression decreased calretinin expression levels in MM cells. The regulation was found to operate bi-directionally, i.e. calretinin overexpression also decreased septin 7 levels. During murine embryonic development calretinin and septin 7 were found to be co-expressed in embryonic mesenchyme and undifferentiated mesothelial cells. In MM cells, calretinin and septin 7 colocalized during cytokinesis in distinct regions of the cleavage furrow and in the midbody region of mitotic cells. Co-immunoprecipitation experiments revealed this co-localization to be the result of a direct interaction between calretinin and septin 7. CONCLUSIONS: Our results demonstrate septin 7 not only serving as a "cytoskeletal" protein, but also as a transcription factor repressing calretinin expression. The negative regulation of calretinin by septin 7 and vice versa sheds new light on mechanisms possibly implicated in MM formation and identifies these proteins as transcriptional regulators and putative targets for MM therapy.

Biological noise and positional effects influence cell stemness.

Biological (or cellular) noise is the random quantitative variability of proteins and other molecules in individual, genetically identical cells. As the result of biological noise in the levels of some transcription factors that determine a cell's differentiation status, differentiated cells may dedifferentiate to a stem cell state given a sufficiently long time period. Here, to provide direct evidence supporting this hypothesis, we used a live-cell monitoring system based on enhanced green fluorescent protein (eGFP) expression to continuously assess the "stemness" of individual human and murine malignant mesothelioma cells over a period of up to 3 months. Re-expression of the transcription factors, the top hierarchical stemness markers Sox2 (SRY-box 2) and Oct4 (octamer-binding transcription factor), monitored as cell eGFP expression was observed in a subpopulation of differentiated eGFP(-) malignant mesothelioma cells. However, we found that this transition was extremely rare. Of note, when it did occur, neighboring cells that were not direct descendants of a newly emerged eGFP(+) stem cell were more likely than non-neighboring cells to also become an eGFP(+) stem cell. This observation suggested a positional effect and led to a clustered "mosaic" reappearance of eGFP(+) stem cells. Moreover, stem cells reappeared even in cell cultures derived from one single differentiated eGFP(-) cell. On the basis of our experimental in vitro and in vivo findings, we developed a tumor growth model to predict the clustered localization of cancer stem cells within a tumor mass.

Menthol evokes Ca2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells.

Menthol is a naturally occurring monoterpene alcohol possessing remarkable biological properties including antipruritic, analgesic, antiseptic, anti-inflammatory and cooling effects. Here, we examined the menthol-evoked Ca2+ signals in breast and prostate cancer cell lines. The effect of menthol (50-500µM) was predicted to be mediated by the transient receptor potential ion channel melastatin subtype 8 (TRPM8). However, the intensity of menthol-evoked Ca2+ signals did not correlate with the expression levels of TRPM8 in breast and prostate cancer cells indicating a TRPM8-independent signaling pathway. Menthol-evoked Ca2+ signals were analyzed in detail in Du 145 prostate cancer cells, as well as in CRISPR/Cas9 TRPM8-knockout Du 145 cells. Menthol (500µM) induced Ca2+ oscillations in both cell lines, thus independent of TRPM8, which were however dependent on the production of inositol trisphosphate. Results based on pharmacological tools point to an involvement of the purinergic pathway in menthol-evoked Ca2+ responses. Finally, menthol (50-500µM) decreased cell viability and induced oxidative stress independently of the presence of TRPM8 channels, despite that temperature-evoked TRPM8-mediated inward currents were significantly decreased in TRPM8-knockout Du 145 cells compared to wild type Du 145 cells.

Pellitorine, an extract of Tetradium daniellii, is an antagonist of the ion channel TRPV1.

BACKGROUND: Transient Receptor Potential Vanilloid 1 (TRPV1) confers noxious heat and inflammatory pain signals in the peripheral nervous system. Clinical trial of resiniferatoxin from Euphorbia species is successfully aimed at TRPV1 in cancer pain management and heading toward new selective painkiller status that further validates this target for drug discovery efforts. Evodia species, used in traditional medicine for hundreds of years, are a recognised source of different TRPV1 agonists, but no antagonist has yet been reported. HYPOTHESIS/PURPOSE: In a search for painkiller leads, we noted for the first time a TRPV1 antagonist activity in the fresh fruits of Tetradium daniellii (Benn.) T.G. Hartley (syn. Evodia hupehensis Dode). METHODS: Through a combination of extraction and purification methods with functional TRPV1-specific Ca2+ uptake assays (bioactivity-guided fractionation/isolation/purification); we isolated a new painkiller candidate that is a distant structural homologue of capsiate exovanilloids and endovanilloids such as anandamide, but a putative competitive inhibitor of the TRPV1. Four additional inactive compounds (N-isobutyl-4,5-epoxy-2E-decadienamide, geranylpsoralen, 8-(7',8'-epoxygeranyloxy)psoralen, and xanthotoxol) were also co-purified with pellitorine. Their structures were established by extensive 1D- and 2D-NMR spectroscopic analysis. RESULTS: 1H- and 13C NMR determination of the chemical structure revealed it to be pellitorine, (2E,4E)-N-(2-methylpropyl)deca-2,4-dienamide, which can compete structurally with algesics released in inflammation. In contrast to previous isolates from Evodia species, pellitorine blocked capsaicin-evoked Ca2+ uptake with an IC50 of 154 µg/ml (0.69 mM/l). N-Isobutyl-4,5-epoxy-2E-decadienamide and geranylpsoralen, 8-(7',8'-epoxygeranyloxy)psoralen, and xanthotoxol did not affect the TRPV1. CONCLUSION: This is the first evidence that pellitorine, an aliphatic alkylamide analogue of capsaicin, can serve as an antagonist of the TRPV1 and may inhibit exovanilloid-induced pain.

Molecular Surgery Concept from Bench to Bedside: A Focus on TRPV1+ Pain-Sensing Neurons.

"Molecular neurosurgery" is emerging as a new medical concept, and is the combination of two partners: (i) a molecular neurosurgery agent, and (ii) the cognate receptor whose activation results in the selective elimination of a specific subset of neurons in which this receptor is endogenously expressed. In general, a molecular surgery agent is a selective and potent ligand, and the target is a specific cell type whose elimination is desired through the molecular surgery procedure. These target cells have the highest innate sensitivity to the molecular surgery agent usually due to the highest receptor density being in their plasma membrane. The interaction between the ligand and its receptor evokes an overactivity of the receptor. If the receptor is a ligand-activated non-selective cation channel, the overactivity of receptor leads to excess Ca2+ and Na+ influx into the cell and finally cell death. One of the best known examples of such an interaction is the effect of ultrapotent vanilloids on TRPV1-expressing pain-sensing neurons. One intrathecal resiniferatoxin (RTX) dose allows for the receptor-mediated removal of TRPV1+ neurons from the peripheral nervous system. The TRPV1 receptor-mediated ion influx induces necrotic processes, but only in pain-sensing neurons, and usually within an hour. Besides that, target-specific apoptotic processes are also induced. Thus, as a nano-surgery scalpel, RTX removes the neurons responsible for generating pain and inflammation from the peripheral nervous system providing an option in clinical management for the treatment of morphine-insensitive pain conditions. In the future, the molecular surgery concept can also be exploited in cancer research for selectively targeting the specific tumor cell.

Targeting breast cancer cells by MRS1477, a positive allosteric modulator of TRPV1 channels.

There is convincing epidemiological and experimental evidence that capsaicin, a potent natural transient receptor potential cation channel vanilloid member 1 (TRPV1) agonist, has anticancer activity. However, capsaicin cannot be given systemically in large doses, because of its induction of acute pain and neurological inflammation. MRS1477, a dihydropyridine derivative acts as a positive allosteric modulator of TRPV1, if added together with capsaicin, but is ineffective, if given alone. Addition of MRS1477 evoked Ca2+ signals in MCF7 breast cancer cells, but not in primary breast epithelial cells. This indicates that MCF7 cells not only express functional TRPV1 channels, but also produce endogenous TRPV1 agonists. We investigated the effects of MRS1477 and capsaicin on cell viability, caspase-3 and -9 activities and reactive oxygen species production in MCF7 cells. The fraction of apoptotic cells was increased after 3 days incubation with capsaicin (10 µM) paralleled by increased reactive oxygen species production and caspase activity. These effects were even more pronounced, when cells were incubated with MRS1477 (2 µM) either alone or together with CAPS (10 µM). Capsazepine, a TRPV1 blocker, inhibited both the effect of capsaicin and MRS1477. Whole-cell patch clamp recordings revealed that capsaicin-evoked TRPV1-mediated current density levels were increased after 3 days incubation with MRS1477 (2 µM). However, the tumor growth in MCF7 tumor-bearing immunodeficient mice was not significantly decreased after treatment with MRS1477 (10 mg/ kg body weight, i.p., injection twice a week). In conclusion, in view of a putative in vivo treatment with MRS1477 or similar compounds further optimization is required.

Stem Cell Factor-Based Identification and Functional Properties of In Vitro-Selected Subpopulations of Malignant Mesothelioma Cells.

Malignant mesothelioma (MM) is an aggressive neoplasm characterized by a poor patient survival rate, because of rapid tumor recurrence following first-line therapy. Cancer stem cells (CSCs) are assumed to be responsible for initiating tumorigenesis and driving relapse after therapeutic interventions. CSC-enriched MM cell subpopulations were identified by an OCT4/SOX2 reporter approach and were characterized by (1) increased resistance to cisplatin, (2) increased sensitivity toward the FAK inhibitor VS-6063 in vitro, and (3) a higher tumor-initiating capacity in vivo in orthotopic xenograft and allograft mouse models. Overexpression of NF2 (neurofibromatosis 2, merlin), a tumor suppressor often mutated or lost in MM, did not affect proliferation and viability of CSC-enriched MM populations but robustly decreased the viability of reporter-negative cells. In contrast, downregulation of calretinin strongly decreased proliferation and viability of both populations. In summary, we have enriched and characterized a small MM cell subpopulation that bears the expected CSC characteristics.

Endogenous TRPV1 stimulation leads to the activation of the inositol phospholipid pathway necessary for sustained Ca2+ oscillations.

Sensory neuron subpopulations as well as breast and prostate cancer cells express functional transient receptor potential vanilloid type 1 (TRPV1) ion channels; however little is known how TRPV1 activation leads to biological responses. Agonist-induced activation of TRPV1 resulted in specific spatiotemporal patterns of cytoplasmic Ca2+ signals in breast and prostate cancer-derived cells. Capsaicin (CAPS; 50µM) evoked intracellular Ca2+ oscillations and/or intercellular Ca2+ waves in all cell lines. As evidenced in prostate cancer Du 145 cells, oscillations were largely dependent on the expression of functional TRPV1 channels in the plasma membrane, phospholipase C activation and on the presence of extracellular Ca2+ ions. Concomitant oscillations of the mitochondrial matrix Ca2+ concentration resulted in mitochondria energization evidenced by increased ATP production. CAPS-induced Ca2+ oscillations also occurred in a subset of sensory neurons, yet already at lower CAPS concentrations (1µM). Stimulation of ectopically expressed TRPV1 channels in CAPS-insensitive NIH-3T3 cells didn't provoke CAPS-triggered Ca2+ oscillations; rather it resulted in low-magnitude, long-lasting elevations of the cytosolic Ca2+ concentration. This indicates that sole TRPV1 activation is not sufficient to generate Ca2+ oscillations. Instead the initial TRPV1-mediated signal leads to the activation of the inositol phospholipid pathway. This in turn suffices to generate a biologically relevant frequency-modulated Ca2+ signal.

Activation of endogenous TRPV1 fails to induce overstimulation-based cytotoxicity in breast and prostate cancer cells but not in pain-sensing neurons.

Vanilloids including capsaicin and resiniferatoxin are potent transient receptor potential vanilloid type 1 (TRPV1) agonists. TRPV1 overstimulation selectively ablates capsaicin-sensitive sensory neurons in animal models in vivo. The cytotoxic mechanisms are based on strong Na(+) and Ca(2+) influx via TRPV1 channels, which leads to mitochondrial Ca(2+) accumulation and necrotic cell swelling. Increased TRPV1 expression levels are also observed in breast and prostate cancer and derived cell lines. Here, we examined whether potent agonist-induced overstimulation mediated by TRPV1 might represent a means for the eradication of prostate carcinoma (PC-3, Du 145, LNCaP) and breast cancer (MCF7, MDA-MB-231, BT-474) cells in vitro. While rat sensory neurons were highly vanilloid-sensitive, normal rat prostate epithelial cells were resistant in vivo. We found TRPV1 to be expressed in all cancer cell lines at mRNA and protein levels, yet protein expression levels were significantly lower compared to sensory neurons. Treatment of all human carcinoma cell lines with capsaicin didn't lead to overstimulation cytotoxicity in vitro. We assume that the low vanilloid-sensitivity of prostate and breast cancer cells is associated with low expression levels of TRPV1, since ectopic TRPV1 expression rendered them susceptible to the cytotoxic effect of vanilloids evidenced by plateau-type Ca(2+) signals, mitochondrial Ca(2+) accumulation and Na(+)- and Ca(2+)-dependent membrane disorganization. Moreover, long-term monitoring revealed that merely the ectopic expression of TRPV1 stopped cell proliferation and often induced apoptotic processes via strong activation of caspase-3 activity. Our results indicate that specific targeting of TRPV1 function remains a putative strategy for cancer treatment.