Transient Receptor Potential Ankyrin 1 Ion Channel Is Expressed in Osteosarcoma and Its Activation Reduces Viability.

Osteosarcoma is a highly malignant, painful cancer with poor treatment opportunities and a bad prognosis. Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors are non-selective cation channels that have been of great interest in cancer, as their expression is increased in some malignancies. In our study we aim to characterize the expression and functionality of the TRPA1 and TRPV1 channels in human and mouse osteosarcoma tissues and in a mouse cell line. TRPA1/Trpa1 and TRPV1/Trpv1 mRNA expressions were demonstrated by PCR gel electrophoresis and RNAscope in situ hybridization. The function of these channels was confirmed by their radioactive 45Ca2+ uptake in response to the TRPA1 agonist, Allyl-isothiocyanate (AITC), and TRPV1 agonist, capsaicin, in K7M2 cells. An ATP-based K2M7 cell viability luminescence assay was used to determine cell viability after AITC or capsaicin treatments. Both TRPA1/Trpa1 and TRPV1/Trpv1 were expressed similarly in human and mouse osteosarcoma tissues, while Trpa1 transcripts were more abundantly present in K7M2 cells. TRPA1 activation with 200 µM AITC induced a significant 45Ca2+ influx into K7M2 cells, and the antagonist attenuated this effect. In accordance with the lower Trpv1 expression, capsaicin induced a moderate 45Ca2+ uptake, which did not reach the level of statistical significance. Both AITC and capsaicin significantly reduced K7M2 cell viability, demonstrating EC50 values of 22 µM and 74 µM. The viability-decreasing effect of AITC was significantly but only partially antagonized by HC-030031, but the action of capsaicin was not affected by the TRPV1 antagonist capsazepine. We provide here the first data on the functional expression of the TRPA1 and TRPV1 ion channels in osteosarcoma, suggesting novel diagnostic and/or therapeutic perspectives.

The heptapeptide somatostatin analogue TT-232 exerts analgesic and anti-inflammatory actions via SST4 receptor activation: In silico, in vitro and in vivo evidence in mice.

Since the conventional and adjuvant analgesics have limited effectiveness frequently accompanied by serious side effects, development of novel, potent pain killers for chronic neuropathic and inflammatory pain conditions is a big challenge. Somatostatin (SS) regulates endocrine, vascular, immune and neuronal functions, cell proliferation through 5 Gi protein-coupled receptors (SST1-SST5). SS released from the capsaicin-sensitive peptidergic sensory nerves mediates anti-inflammatory and antinociceptive effects without endocrine actions via SST4. The therapeutic use of the native SS is limited by its diverse biological actions and short plasma elimination half-life. Therefore, SST4 selective SS analogues could be promising analgesic and anti-inflammatory drug candidates with new mode of action. TT-232 is a cyclic heptapeptide showing great affinity to SST4 and SST1. Here, we report the in silico SST4 receptor binding mechanism, in vitro binding (competition assay) and cAMP- decreasing effect of TT-232 in SST4-expressing CHO cells, as well as its analgesic and anti-inflammatory actions in chronic neuropathic pain and arthritis models using wildtype and SST4-deficient mice. TT-232 binds to SST4 with similar interaction energy (-11.03 kcal/mol) to the superagonist J-2156, displaces somatostatin from SST4 binding (10 nM to 30 µM) and inhibits forskolin-stimulated cAMP accumulation (EC50: 371.6 ± 58.03 nmol; Emax: 78.63 ± 2.636 %). Its i.p. injection (100, 200 µg/kg) results in significant, 35.7 % and 50.4 %, analgesic effects upon single administration in chronic neuropathic pain and repeated injection in arthritis models in wildtype, but not in SST4-deficient mice. These results provide evidence that the analgesic effect of TT-232 is mediated by SST4 activation, which might open novel drug developmental potentials. Chemical compounds Chemical compounds studied in this article TT-232 (PubChem CID: 74053735).

Synthetic Diphenylacetylene-Based Retinoids Induce DNA Damage in Chinese Hamster Ovary Cells without Altering Viability.

All-trans-retinoic acid (ATRA), the active metabolite of vitamin A, plays a pivotal role in cell differentiation, proliferation and embryonic development. It is an effective therapy for dermatological disorders and malignancies. ATRA is prone to isomerization and oxidation, which can affect its activity and selectivity. Novel diphenylacetylene-based ATRA analogues with increased stability can help to overcome these problems and may offer significant potential as therapeutics for a variety of cancers and neurodegenerative diseases, including amyotrophic lateral sclerosis. Here, we investigated the effects of these retinoids on cell viability and genotoxicity in the widely used model system of the rapidly proliferating Chinese hamster ovary cell line. DC360 is a fluorescent ATRA analogue and DC324 is a non-active derivative of DC360. EC23, DC525, DC540, DC645, and DC712 are promising analogues with increased bioactivity. The cytotoxic activity of the compounds was evaluated by ATP assay and DNA damage was tested by comet assay. No cytotoxicity was observed in the 10-6-10-5 M concentration range. All compounds induced DNA migration similar to ATRA, but DC324, DC360 and EC23 did so to a greater extent, particularly at higher concentrations. We believe that retinoid receptor-independent genotoxicity is a general characteristic of these compounds; however, further studies are needed to identify the molecular mechanisms and understand their complex biological functions.

In Silico, In Vitro and In Vivo Pharmacodynamic Characterization of Novel Analgesic Drug Candidate Somatostatin SST4 Receptor Agonists.

Background: Somatostatin released from the capsaicin-sensitive sensory nerves mediates analgesic and anti-inflammatory effects via its receptor subtype 4 (SST4) without influencing endocrine functions. Therefore, SST4 is considered to be a novel target for drug development in pain, especially chronic neuropathy which is a great unmet medical need. Purpose and Experimental Approach: Here, we examined the in silico binding, SST4-linked G protein activation and ß-arrestin activation on stable SST4 expressing cells and the effects of our novel pyrrolo-pyrimidine molecules (20, 100, 500, 1,000, 2,000 µg·kg-1) on partial sciatic nerve ligation-induced traumatic mononeuropathic pain model in mice. Key Results: The novel compounds bind to the high affinity binding site of SST4 the receptor and activate the G protein. However, unlike the reference SST4 agonists NNC 26-9100 and J-2156, they do not induce ß-arrestin activation responsible for receptor desensitization and internalization upon chronic use. They exert 65-80% maximal anti-hyperalgesic effects in the neuropathy model 1 h after a single oral administration of 100-500 µg·kg-1 doses. Conclusion and Implications: The novel orally active compounds show potent and effective SST4 receptor agonism in vitro and in vivo. All four novel ligands proved to be full agonists based on G protein activation, but failed to recruit ß-arrestin. Based on their potent antinociceptive effect in the neuropathic pain model following a single oral administration, they are promising candidates for drug development.