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.

SummaryChronic exposure to intraperitoneal asbestos triggered a marked response in the mesothelium well before tumor development. Macrophages, mesothelial precursor cells, cytokines and growth factors accumulated in the peritoneal lavage. Transcriptome profiling revealed YAP/TAZ activation in inflamed mesothelium with further activation in tumors, paralleled by increased levels of cells with nuclear YAP/TAZ. Arg1 was one of the highest upregulated genes in inflamed tissue and tumor. Inflamed tissue showed increased levels of single nucleotide variations, with an RNA-editing signature, which were even higher in the tumor samples. Subcutaneous injection of asbestos-treated, but tumor-free mice with syngeneic mesothelioma tumor cells resulted in a significantly higher incidence of tumor growth when compared to naïve mice supporting the role of the environment in tumor progression.

BackgroundUse of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects.MethodsTo address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies.ResultsAlthough both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1–50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca2+-cytotoxity ([RTX]>15 µM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes.ConclusionTRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials.

234Mammalian temperature sensors of the transient receptor potential (TRP) family are tetrameric 6-transmembrane cation channels gated by heat, cold, as well as either endogenous or exogenous agonists 1,2 . The vanilloid receptor (VR1/TRPV1) is a non-selective ligand-gated Na + /Ca 2+ -channel [3][4][5][6] . TRPV1 is selectively activated by heat above 42ºC, pH < 6.3 7 , endogenous lipid mediators such as anandamide and oleoylethanolamide 4,8,9 , intracellular signaling molecules 10,11 and plant toxins, including capsaicin (CAPS) and resiniferatoxin (RTX).TRPA1 has recently been suggested to serve as receptor of noxious cold temperature 12 , and it transduces pain induced by ABSTRACT: Objectives: Resiniferatoxin, the most potent agonist of inflammatory pain/vanilloid receptor/cation channel (TRPV1) can be used for neuron subtype specific ablation of pain generating cells at the level of the peripheral nervous system by Ca 2+ -excytotoxicity. Molecular neurosurgery is an emerging technology either to alleviate severe pain in cancer or treat/prevent different local neuropathies. Our aim was determining sensory modalities that may be lost after resiniferatoxin treatment. Methods: Newborn or adult mice were treated with resiniferatoxin, then changes in chemical and heat sensitivity were correlated with alterations of the cell composition of sensory ganglions. Results: Only mice treated at adult age became less sensitive to heat stimuli, while both treatment groups lost sensitivity to specific vanilloid agonists of TRPV1 and, interestingly, to allyl-isothiocyanate, a selective agonist of TRPA1. Our in vivo and post mortem analytical results confirmed that TRPV1 and TRPA1 function together and resiniferatoxin-mediated neurosurgery removes both sensor molecules. Discussion: In adult mice resiniferatoxin causes: i) desensitization to heat and ii) sensitization to cold. Cold hyperalgesia, an imbalance in thermosensation, might be conferred by a prominent cold receptor that is expressed in surviving resiniferatoxin-resistant sensory neurons and compensates for pain signals lost with TRPA1 and TRPV1 double positive cells in the peripheral nervous system. RÉSUMÉ: L'ablation de neurones TRPV1+ par la résinifératoxine élimine aussi les neurones TRPA1.Objectifs : La résinifératoxine, l'agoniste le plus puissant de la douleur inflammatoire/du récepteur vanilloïde/du canal cationique (TRPV1), peut être utilisée pour l'ablation spécifique par excytotoxicité Ca2+, d'un sous-type de neurones faisant partie des cellules génératrices de douleur au niveau du système nerveux périphérique. La neurochirurgie moléculaire est une technologie émergente, pour soulager la douleur cancéreuse sévère ou pour traiter ou prévenir différentes neuropathies locales. Notre but était de déterminer les pertes sensitives suite au traitement par la résinifératoxine. Méthodes : Des souris ont été traitées par la résinifératoxine à la période néonatale ou adulte et les changements de la sensibilité chimique et calorique ont été corrélés aux altérations de l...

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.

Mesothelial cells are susceptible to asbestos fiber-induced cytotoxicity and on longer time scales to transformation; the resulting mesothelioma is a highly aggressive neoplasm that is considered as incurable at the present time Zucali et al. (Cancer Treatment Reviews 37:543–558, 2011). Only few murine cell culture models of immortalized mesothelial cells and mesothelioma cell lines exist to date. We generated SV40-immortalized cell lines derived from wild-type (WT) and neurofibromatosis 2 (merlin) heterozygote (Nf2+/−) mice, both on a commonly used genetic background, C57Bl/6J. All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum. Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate. The tumor suppressor gene NF2 is one of the most frequently mutated genes in human mesothelioma, but its detailed function is still unknown. Thus, these genotypically distinct cell lines likely relevant for malignant mesothelioma formation are expected to serve as useful in vitro models, in particular to compare with in vivo studies in mice of the same genotype. Furthermore, we generated a novel murine mesothelioma cell line RN5 originating from an Nf2+/− mouse subjected to repeated crocidolite exposure. RN5 cells are highly tumorigenic.

SummaryMalignant 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.

Transient receptor potential vanilloid subtype 1 (TRPV1) receptor is a pain-sensing, ligand-gated, non-selective cation channel expressed in peripheral sensory neurons. Prolonged activation of TRPV1 by capsaicin leads to cell swelling and formation of membrane blebs in rat dorsal root ganglion (DRG) neurons. Similar results were obtained in NIH3T3 fibroblast cells stably expressing TRPV1. Here, we assessed the contribution of Ca(2+) and Na(+) ions to TRPV1-mediated changes. Cell swelling was caused by a substantial influx of extracellular Na(+) via TRPV1 channels, causing concomitant transport of water. In the absence of extracellular Na(+), the membrane blebbing was completely inhibited, but Ca(2+) influx did not change under these conditions. Na(+) influx was modulated by the intracellular Ca(2+) concentration ([Ca(2+)]i). Elevation of [Ca(2+)]i by ionomycin sensitized/activated TRPV1 channels causing cell swelling in TRPV1-positive cells. In the absence of extracellular Ca(2+), capsaicin caused only little increase in [Ca(2+)]i indicating that the increase in [Ca(2+)]i observed after capsaicin application is derived essentially from extracellular Ca(2+) and not from internal Ca(2+) stores. In the absence of extracellular Ca(2+) also the process of cell swelling was considerably slower. Calretinin is a Ca(2+) buffer protein, which is expressed in a subset of TRPV1-positive neurons. Calretinin decreased the amplitude, but slowed down the decay of Ca(2+) signals evoked by ionomycin. Cells co-expressing TRPV1 and calretinin were less sensitive to TRPV1-mediated, capsaicin-induced volume increases. In TRPV1-expressing NIH3T3 cells, calretinin decreased the capsaicin-induced Ca(2+) and Na(+) influx. Swelling and formation of membrane blebs resulted in impaired plasma membrane integrity finally leading to cell death. Our results hint towards a mechanistic explanation for the apoptosis-independent capsaicin-evoked neuronal loss and additionally reveal a protective effect of calretinin; we propose that the Ca(2+)-buffering capacity of calretinin reduces the susceptibility of calretinin-expressing DRG neurons against cell swelling/death caused by overstimulation of TRPV1 channels. This article is part of a Special Issue entitled:12th European Symposium on Calcium.