Utjecaj osteogeničnih dodataka agregatu mineralnog trioksida na razinu genske ekspresije odontoblastičnih markera nakon zatvaranja pulpe u pasa.

This study investigates the effect of addition of dexamethasone, vitamin D3, or chitosan to mineral trioxide aggregate (MTA) on the gene expression level of dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE) after pulp capping in dogs. Pulp exposure was performed in sixty dogs’ teeth. The teeth were classified into 3 equal groups according to the evaluation period. Group 1:7 days, group 2: 21 days and group 3: 60 days. Each group was further subdivided according to the pulp capping material used, into subgroup A: MTA + dexamethasone, subgroup B: MTA + dexamethasone + vitamin D3, subgroup C: MTA + chitosan and subgroup D: MTA. According to the group, the pulps of the capped teeth were removed for analysis of the relative mRNA expression level of DSPP and MEPE using PCR. Statistical analysis of all data was performed. In subgroup A, significant expression was observed of DSPP (P≤0.05) in group 2 up to 18.8 relative fold change while in subgroup B a significant upregulated gene expression of DSPP (P≤0.05) up to 29.4 relative fold change was seen. Significant upregulated DSPP expression (P≤0.05) was recorded in groups 1 and 2 up to 6.9 and 3.6 relative fold change, respectively in subgroup C. In conclusion, dexamethasone, with or without vitamin D3 and chitosan, are synergistic odontogenic inducers with MTA for differentiation of dental pulp cells in dogs. The upregulation of DSPP is a good marker for this differentiation.Istraživan je utjecaj dodatka deksametazona, vitamina D3 ili hitozana agregatu mineralnog trioksida na razinu ekspresije gena za dentin-sijalofosfoprotein (DSPP) i gena za ekstracelularni matriks fosfoglikoprotein nakon zatvaranja pulpe u pasa. Pulpa je bila otvorena na 60 zuba. Ti su zubi bili razvrstani u tri jednake skupine ovisno o trajanju promatranja. Prva skupina bila je promatrana sedam dana, druga skupina 21 dan, a treća skupina 60 dana. Svaka skupina bila je podijeljena u podskupine ovisno o materijalu rabljenom za zatvaranje pulpe. Podskupini A bio je primijenjen agregat mineralnog trioksida + deksametazon, podskupini B bio je primijenjen agregat mineralnog trioksida + deksametazon + vitamin D3, podskupini C agregat mineralnog trioksida + hitozan, a podskupini D samo agregat mineralnog trioksida. Sukladno skupinama, pulpe zatvorenih zuba bile su uzete za analizu relativne ekspresije mRNA za dentin-sijalofosfoprotein i za ekstracelularni matriks fosfoglikoprotein PCR-om. Svi podatci bili su statistički obrađeni. Značajna ekspresija gena za dentinsijalofosfoprotein (P≤0,05) bila je dokazana u podskupini A druge skupine u relativnoj vrijednosti 18,8, dok je u podskupini B značajna ekspresija gena za dentin-sijalofosfoprotein (P≤0,05) bila 29,4 puta veća. Značajno povećana ekspresija DSPP (P≤0,05) u prvoj i drugoj skupini sve do 6,9 odnosno 3,6 puta ustanovljena je u podskupini C. Može se zaključiti da deksametazon (s vitaminom D3 ili bez njega) i hitozan djeluju sinergistički s agregatom mineralnog trioksida kao odontogenični pokretači za diferencijaciju stanica zubne pulpe u pasa. Povećana razina dentin-sijalofosfoproteina dobar je pokazatelj te diferencijacije

Dual mTORC1/mTORC2 blocker as a possible therapy for tauopathy in cellular model

Tauopathy comprises a group of disorders caused by abnormal aggregates of tau protein. In these disorders phosphorylated tau protein tends to accumulate inside neuronal cells (soma) instead of the normal axonal distribution of tau. A suggested therapeutic strategy for tauopathy is to induce autophagy to increase the ability to get rid of the unwanted tau aggregates. One of the key controllers of autophagy is mTOR. Blocking mTOR leads to stimulation of autophagy. Recently, unravelling molecular structure of mTOR showed that it is formed of two subunits: mTORC1/C2. So, blocking both subunits of mTOR seems more attractive as it will explore all abilities of mTOR molecule. In the present study, we report using pp242 which is a dual mTORC1/C2 blocker in cellular model of tauopathy using LUHMES cell line. Adding fenazaquin to LUHMES cells induced tauopathy in the form of increased phospho tau aggregates. Moreover, fenazaquin treated cells showed the characteristic somatic redistribution of tau. PP242 use in the present tauopathy model reversed the pathology significantly without observable cellular toxicity for the used dosage of 1000 nM. The present study suggests the possible use of pp242 as a dual mTOR blocker to treat tauopathy

Genetic Interpretation of the Impacts of Honokiol and EGCG on Apoptotic and Self-Renewal Pathways in HEp-2 Human Laryngeal CD44\u3csup\u3ehigh\u3c/sup\u3e Cancer Stem Cells

Most current larynx cancer therapies are generally aimed at the global mass of tumor, targeting the non-tumorigenic cells, and unfortunately sparing the tumorigenic cancer stem cells (CSCs) that are responsible for sustained growth, metastasis, and chemo- and radioresistance. Phytochemicals and herbs have recently been introduced as therapeutic sources for eliminating CSCs. Therefore, we assessed the anti-tumor effects of two herbal ingredients, the green tea extract “Epigallocatechin-3-gallate (EGCG)” and Honokiol (HNK), on parental cells or CD44high CSCs of the human laryngeal squamous cell carcinoma cell line HEp-2. Results revealed that EGCG had a preeminent apoptotic potential on HEp-2 laryngeal CSCs. HNK conferred higher cytotoxic impacts on parental cells mostly by necrosis induction, especially with higher doses, but apoptosis induction with lower doses was also observed. The Notch signaling pathway genes were more potently suppressed by EGCG than HNK. However, HNK surpassed EGCG in downregulating the β-catenin and the Sonic Hedgehog signaling pathways genes. On a genetic basis, both agents engaged the BCL-2 family-regulated and caspase-dependent intrinsic apoptotic pathway, but EGCG and HNK triggered apoptosis via p53-independent and p53-dependent pathways, respectively. Taken together, EGCG and HNK eradicated HEp-2 human larynx cancer cells through targeting multiple self-renewal pathways and activating diverse cell death modalities

Design, synthesis, and biological characterization of proteolysis targeting chimera (PROTACs) for the Ataxia telangiectasia and RAD3-related (ATR) kinase

The Ataxia telangiectasia and RAD3-related (ATR) kinase is a key regulator of DNA replication stress responses and DNA-damage checkpoints. Several potent and selective ATR inhibitors are reported and four of them are currently in clinical trials in combination with radio- or chemotherapy. Based on the idea of degrading target proteins rather than inhibiting them, we designed, synthesized and biologically characterized a library of ATR-targeted proteolysis targeting chimera (PROTACs). Among the synthesized compounds, the lenalidomide-based PROTAC 42i was the most promising. In pancreatic and cervix cancer cells cancer cells (MIA PaCa-2), it reduced ATR to 40% of the levels in untreated cells. 42i selectively degraded ATR through the proteasome, dependent on the E3 ubiquitin ligase component cereblon, and without affecting the associated kinases ATM and DNA-PKcs. 42i may be a promising candidate for further optimization and biological characterization in various cancer cells

Novel hydroxamic acid derivative induces apoptosis and constrains autophagy in leukemic cells

Introduction: Posttranslational modification of proteins by reversible acetylation regulates key biological processes. Histone deacetylases (HDACs) catalyze protein deacetylation and are frequently dysregulated in tumors. This has spurred the development of HDAC inhibitors (HDACi). Such epigenetic drugs modulate protein acetylation, eliminate tumor cells, and are approved for the treatment of blood cancers. Objectives: We aimed to identify novel, nanomolar HDACi with increased potency over existing agents and selectivity for the cancer-relevant class I HDACs (HDAC1,-2,-3,-8). Moreover, we wanted to define how such drugs control the apoptosis-autophagy interplay. As test systems, we used human leukemic cells and embryonic kidney-derived cells. Methods: We synthesized novel pyrimidine-hydroxamic acid HDACi (KH9/KH16/KH29) and performed in vitro activity assays and molecular modeling of their direct binding to HDACs. We analyzed how these HDACi affect leukemic cell fate, acetylation, and protein expression with flow cytometry and immunoblot. The publicly available DepMap database of CRISPR-Cas9 screenings was used to determine sensitivity factors across human leukemic cells. Results: Novel HDACi show nanomolar activity against class I HDACs. These agents are superior to the clinically used hydroxamic acid HDACi SAHA (vorinostat). Within the KH-series of compounds, KH16 (yanostat) is the most effective inhibitor of HDAC3 (IC50 = 6 nM) and the most potent inducer of apoptosis (IC50 = 110 nM; p < 0.0001) in leukemic cells. KH16 though spares embryonic kidney-derived cells. Global data analyses of knockout screenings verify that HDAC3 is a dependency factor in 115 human blood cancer cells of different lineages, independent of mutations in the tumor suppressor p53. KH16 alters pro- and anti-apoptotic protein expression, stalls cell cycle progression, and induces caspase-dependent processing of the autophagy proteins ULK1 and p62. Conclusion: These data reveal that HDACs are required to stabilize autophagy proteins through suppression of apoptosis in leukemic cells. HDAC3 appears as a valid anti-cancer target for pharmacological intervention