Synthesis, in vitro and in vivo evaluation of 1,3,5-triazines as cannabinoid CB2 receptor agonists

The cannabinoid receptors type 2 (CBR2) are attractive therapeutic targets of the endocannabinoid signaling system (ECS) as they are not displaying the undesired psychotropic and cardiovascular side-effects seen with cannabinoid receptor type 1 (CB1R) agonists. In continuation of our previous work on 2,4,6-trisubstituted 1,3,5-triazines as potent CB2 agonists, we synthesized an additional series of more polar analogues (1-10), which were found to possess high CB2R agonist activity with enhanced water solubility. The most potent compound in the series was N-(adamantan-1-yl)-4-ethoxy-6-(4-(2-fluoroethyl)piperazin-1-yl)-1,3,5-triazin-2-amine (9) with EC50 value of 0.60nM. To further evaluate the biological effects of the compounds, the selected compounds were tested in vitro against four different cell lines. A human retinal pigment epithelial cell line (ARPE-19) was used to evaluate the cytotoxicity of the compounds whereas an androgen-sensitive human prostate adenocarcinoma cell line (LNCaP), a Jurkat leukemia cell line and a C8161 melanoma cell line were used to assess the antiproliferative activity of the compounds. The most interesting results were obtained for N-(adamantan-1-yl)-4-ethoxy-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (6), which induced cell viability decrease in prostate and leukemia cell lines, and diminished proliferation of C8161 melanoma cells. The results could be reversed in leukemia cells with the selective CB2R antagonist AM630, whereas in prostate cells the AM630 induced a significant cell viability decrease with a mechanism probably unlinked to CB2 cannabinoid receptor. The antiproliferative effect of 6 on the melanoma cells seemed not to be mediated via the CB1R or CB2R. No cytotoxicity was detected against ARPE-19 cell line at concentrations of 1 and 10μM for compound 6. However, at 30μM concentration the compound 6 decreased the cell viability. Finally, in order to estimate in vivo behavior of these compounds, (18)F labeled PET ligand, N-cyclopentyl-4-ethoxy-6-(4-(2-fluoro-18-ethyl)piperazin-1-yl)-1,3,5-triazin-2-amine ([(18)F]5), was synthesized and its biodistribution was determined in healthy male Sprague-Dawley rats. As a result, the tracer showed a rapid (<15min) elimination in urine accompanied by a slower excretion via the hepatobiliary route. In conclusion, we further demonstrated that 1,3,5-triazine scaffold serves as a suitable template for the design of highly potent CB2R agonists with reasonable water solubility properties. The compounds may be useful when studying the role of the endocannabinoid system in different diseases. The triazine scaffold is also a promising candidate for the development of new CB2R PET ligands

Metformin decreases hyaluronan synthesis by vascular smooth muscle cells

Metformin is the first-line drug in the treatment of type 2 diabetes worldwide based on its effectiveness and cardiovascular safety. Currently metformin is increasingly used during pregnancy in women with gestational diabetes mellitus, even if the long-term effects of metformin on offspring are not exactly known. We have previously shown that high glucose concentration increases hyaluronan (HA) production of cultured human vascular smooth muscle cells (VSMC) via stimulating the expression of hyaluronan synthase 2 (HAS2). This offers a potential mechanism whereby hyperglycemia leads to vascular macroangiopathy. In this study, we examined whether gestational metformin use affects HA content in the aortic wall of mouse offspring in vivo. We also examined the effect of metformin on HA synthesis by cultured human VSMCs in vitro. We found that gestational metformin use significantly decreased HA content in the intima-media of mouse offspring aortas. In accordance with this, the synthesis of HA by VSMCs was also significantly decreased in response to treatment with metformin. This decrease in HA synthesis was shown to be due to the reduction of both the expression of HAS2 and the amount of HAS substrates, particularly UDP-N-acetylglucosamine. As shown here, gestational metformin use is capable to program reduced HA content in the vascular wall of the offspring strongly supporting the idea, that metformin possesses long-term vasculoprotective effects.</p

HAS3-induced extracellular vesicles from melanoma cells stimulate IHH mediated c-Myc upregulation via the hedgehog signaling pathway in target cells.

Intercellular communication is fundamental to the survival and maintenance of all multicellular systems, whereas dysregulation of communication pathways can drive cancer progression. Extracellular vesicles (EVs) are mediators of cell-to-cell communication that regulate a variety of cellular processes involved in tumor progression. Overexpression of a specific plasma membrane enzyme, hyaluronan synthase 3 (HAS3), is one of the factors that can induce EV shedding. HAS3, and particularly its product hyaluronan (HA), are carried by EVs and are known to be associated with the tumorigenic properties of cancer cells. To elucidate the specific effects of cancerous, HAS3-induced EVs on target cells, normal human keratinocytes and melanoma cells were treated with EVs derived from GFP-HAS3 expressing metastatic melanoma cells. We found that the HA receptor CD44 participated in the regulation of EV binding to target cells. Furthermore, GFP-HAS3-positive EVs induced HA secretion, proliferation and invasion of target cells. Our results suggest that HAS3-EVs contains increased quantities of IHH, which activates the target cell hedgehog signaling cascade and leads to the activation of c-Myc and regulation of claspin expression. This signaling of IHH in HAS3-EVs resulted in increased cell proliferation. Claspin immunostaining correlated with HA content in human cutaneous melanocytic lesions, supporting our in vitro findings and suggesting a reciprocal regulation between claspin expression and HA synthesis. This study shows for the first time that EVs originating from HAS3 overexpressing cells carry mitogenic signals that induce proliferation and epithelial-to-mesenchymal transition in target cells. The study also identifies a novel feedback regulation between the hedgehog signaling pathway and HA metabolism in melanoma, mediated by EVs carrying HA and IHH.Peer reviewe

Modelling the human epidermis in vitro: tools for basic and applied research

Culture models of tissues and organs are valuable tools developed by basic research that help investigation of the body functions. Modelling is aimed at simplifying experimental procedures in order to better understand biological phenomena, and consequently, when sufficiently characterized, culture models can also be utilized with high potential in applied research. In skin biology and pathology, the development of cultures of keratinocytes as monolayers has allowed the elucidation of most functional and structural characteristics of the cell type. Beside the multiple great successes that have been obtained with this type of culture, this review draws attention on several neglected characteristics of monolayer cultures. The more sophisticated models created in order to reconstruct the fully differentiated epidermis have followed the monolayers. The epidermal reconstruction produces all typical layers found in vivo and thus makes the model much less simple, but only this kind of model allows the study of full differentiation in keratinocyte and production of the cornified barrier. In addition to its interest in basic research, the reconstructed epidermis is currently gaining a lot of interest for applied research, particularly as an alternative to laboratory animals in the chemical and cosmetic industry. Today several commercial providers propose reconstructed skin or epidermis, but in vitro assays on these materials are still under development. In order to be beneficial at long term, the validation of assays must be performed on a material whose availability will not be interrupted. We warn here providers and customers that the longevity of in vitro assays will be guaranteed only if these assays are done with well-described models, prepared according to published procedures, and must consider having a minimum of two independent simultaneous producers of similar material

Cellular Phenotype-Dependent and -Independent Effects of Vitamin C on the Renewal and Gene Expression of Mouse Embryonic Fibroblasts

Vitamin C has been shown to delay the cellular senescence and was considered a candidate for chemoprevention and cancer therapy. To understand the reported contrasting roles of vitamin C: growth-promoting in the primary cells and growth-inhibiting in cancer cells, primary mouse embryonic fibroblasts (MEF) and their isogenic spontaneously immortalized fibroblasts with unlimited cell division potential were used as the model pair. We used microarray gene expression profiling to show that the immortalized MEF possess human cancer gene expression fingerprints including a pattern of up-regulation of inflammatory response-related genes. Using the MEF model, we found that a physiological treatment level of vitamin C (10−5 M), but not other unrelated antioxidants, enhanced cell growth. The growth-promoting effect was associated with a pattern of enhanced expression of cell cycle- and cell division-related genes in both primary and immortalized cells. In the immortalized MEF, physiological treatment levels of vitamin C also enhanced the expression of immortalization-associated genes including a down-regulation of genes in the extracellular matrix functional category. In contrast, confocal immunofluorescence imaging of the primary MEF suggested an increase in collagen IV protein upon vitamin C treatment. Similar to the cancer cells, the growth-inhibitory effect of the redox-active form of vitamin C was preferentially observed in immortalized MEF. All effects of vitamin C required its intracellular presence since the transporter-deficient SVCT2−/− MEF did not respond to vitamin C. SVCT2−/− MEF divided and became immortalized readily indicating little dependence on vitamin C for the cell division. Immortalized SVCT2−/− MEF required higher concentration of vitamin C for the growth inhibition compared to the immortalized wildtype MEF suggesting an intracellular vitamin C toxicity. The relevance of our observation in aging and human cancer prevention was discussed

Jatko-opintojen ohjaushyviä ohjauskäytänteitä jatko-opintojen ohjaajille /

Tutkimus ja viimeisimpään tutkimustietoon perustuva opetus ovat yliopistojen keskeisimmät tehtävät. Suurimman osan yliopistoissa tehtävästä tutkimuksesta tekevät tohtorikoulutettavat omissa väitöskirjaprojekteissaan. Tohtorikoulutettavat vievät tiedettä eteenpäin Suomessa ja juuri heidän tutkimustulostensa ja valmistuvien väitöskirjojen (tutkintojen) perusteella yliopistot saavat ulkopuolista rahoitusta. Tohtorikoulutettavien työhyvinvointi ja sen myötä tuloksellisuus vaikuttavat hyvin laajasti koko akateemiseen yhteisöön. Opetusministeriö teetti vuonna 2005 tutkimuksen suomalaisesta tohtorikoulutuksesta, jossa selvitettiin jatko-opiskelijoiden kokemuksia ja arvioita koulutuksestaan. Tässä tutkimuksessa selvisi, että tohtorikoulutettavat ovat tyytyväisiä saamaansa jatkokoulutukseen mutta kokevat suuria puutteita jatko-opintojen perehdyttämisessä, ohjauksessa ja mentoroinnissa. Suuri osa koki, ettei ohjaaja ollut osoittanut mielenkiintoa heidän jatko-opintojaan kohtaan ja keskustellut heidän kanssa tutkimukseen liittyvistä asioista. Nämä ongelmat ohjauksessa hidastivat väitöskirjatutkimuksen etenemistä. Tämän kehittämishankkeen tavoitteena on käsitellä jatko-opintojen ohjausprosessia. Hankkeessa kartoitetaan jatko-opintoihin ja niiden ohjaamiseen liittyviä asioita sekä pyritään löytämään ohjausprosessista kriittisiä kohtia, joissa ohjauksen tarve korostuu jotta tohtorikoulutettava saisi tarvitsemaansa tukea väitöskirjatyössään ja ammatillisessa kehittymisessään. Työssä kuvataan jatko-opintojen ohjaamiseen ohjauskäytänteitä, joissa otetaan huomioon opiskelun ohjaus, urasuunnittelun ohjaus ja persoonallisen kasvun tukeminen. On kuitenkin muistettava, että ohjaustyö on aina opiskelijalähtöistä, joten yhtä ainutta oikeaa ohjausmallia ei ole olemassa, sillä jokainen opiskelija on erilainen.The mission of universities is to conduct scientific research and provide undergraduate and postgraduate education based on it. Doctoral students are responsible for most of the research work done in the universities. They promote the science in Finland and universities get resources just based on their research results and doctoral theses (the number of doctor’s degree). The working welfare of doctoral students and thus their profitability affects greatly to the whole academic community. In 2005, the Ministry of Education investigated Finnish doctoral education. This research focused on doctoral students’ experiences and evaluations of their education. In this research, it was found that doctoral students are generally satisfied with the content of postgraduate studies but they have confronted great deficiencies in introducing to the postgraduate studies, in supervision and in all kind of mentoring. Most of doctoral students felt that their supervisor is not interested in their doctoral studies and has not discussed relevant issues related to their research work with them. Their doctoral thesis work was delayed by these problems. The aim of this development project is to deal with processes of student guidance in postgraduate studies. This development project describes the issues related to postgraduate studies and to the supervision of these studies. In this project, it is aspired to find critical phases in doctoral studies where the need of supervision is emphasized. This work aims to describe good supervision practices, in which the study guidance, career supervision and personal growth support are taken into account. However, one must not forget that all kind of supervision must be student-centred and it is impossible to give one supervision model, because all students are different

Altered expression of hyaluronan, HAS1‐2, and HYAL1‐2 in oral lichen planus

Oral lichen planus (OLP) is an immune‐mediated mucosal disease of unclear etiology and of unresolved pathogenesis. Hyaluronan (HA) is an extracellular matrix glycosaminoglycan involved in inflammation and tumor progression. However, its presence in OLP has not been reported. We therefore aimed to study the immunohistochemical expression of HA, its receptor CD44, hyaluronan synthases (HAS1‐3), and hyaluronidases (HYAL1‐2) in OLP. Methods The presence of HA, CD44, HAS1‐3, and HYAL1‐2 was studied by immunohistochemical methods in 55 OLP and 23 control oral mucosal specimens (CTR). The localization, intensity, and differences of the epithelial expression between OLP and CTRs were analyzed. Results HA and CD44 were found on cell membranes in the epithelial basal and intermediate layers in CTR and OLP specimens. The HA staining intensity was stronger in the basal layer of the epithelium in OLP than in CTRs (P < 0.001). HAS1 (P = 0.001) and HAS2 (P < 0.001) showed stronger staining in the basal and weaker staining in the superficial (P < 0.001) epithelial layers in OLP than in CTRs. The immunostaining of HAS3 was low in both OLP and CTRs. Positive HYAL1 and HYAL2 staining were mainly found in the basal and intermediate epithelial layers, and their intensities were significantly increased in OLP, except HYAL 2 in the intermediate epithelial layer. Conclusions HA, HAS1‐2, and HYAL1‐2 have altered expression in OLP compared to CTRs and may therefore have a role in OLP pathogenesis44640140