Structural and functional characterization of HMGB1 protein in rat liver during experimentally-induced diabetes type 1

Оксидативни стрес и хронична инфламација сматрају се главним узроцима појаве дијабетичних компликација, међу којима су и оштећења јетре. Важну улогу медијатора ових процеса може имати ендогени протеин HMGB1, који у ванћелијску средину доспева из некротичних, оштећених и активираних ћелија. Како је у дијабетесу понашање HMGB1 протеина слабо изучавано, у овој докторској дисертацији испитиван је допринос HMGB1 оштећењима јетре пацова са стрептозотоцином-изазваним ДТ1. Показано је да ниво оштећења јетре током дијабетесa корелише са присуством ванћелијског HMGB1. Овај протеин, у дијабетичној јетри, бива структурно модификован ацетилацијом, фосфорилацијом и O-GlcNAc гликозилацијом што корелише са његовим изласком из једра ћелија у цитоплазму и повећањем његовог присуства у јетри и серуму. Резултати у вези са снижавањeм нивоа ванћелијског HMGB1 третманом дијабетичних пацова мелатонином или етил пируватом, указују да HMGB1 доприноси оштећењу јетре у дијабетесу одржавањем стања хроничне инфламације, стишавањем антиоксидативне одбране и стишавањем регенерације. Ванћелијски HMGB1 кроз интеракције са TLR4 рецептором активира MAPК/NF-κB p65 и ЈАК1/STAT3 сигналне путеве, доприносећи повећању продукције проинфламацијских цитокина TNF-α и IL-6 и акутно-фазног протеина хаптоглобина. Подстицањем NF-κB p65 инфламацијског пута, HMGB1 делује негативно на цитопротективни одговор у дијабетичној јетри тако што онемогућава активност Nrf2 протеина, одговорног за стишавање инфламације и продукцију антиоксидативних ензима. На стишавање регенеративног потенцијала јетре, активирана HMGB1/TLR4 оса утиче преко увећања присуства негативних регулатора ћелијског циклуса - протеина p53 и p21, и смањењем нивоа циклина D1. Добијени резултати указују на сложеност деловања HMGB1 протеина у дијабетесу и на значај спречавања ослобађања HMGB1 или блокаде HMGB1/TLR4 осе у циљу одлагања настанка оштећења јетре.Oxidative stress and chronic inflammation are considered to be the main causes of diabetic complications, one of which is liver damage. An important mediator of these processes may be the endogenous HMGB1 protein, when released into the extracellular environment from the necrotic, damaged or activated cells. As the HMGB1 role in diabetes was insufficiently studied, in this doctoral dissertation the contribution of HMGB1 to liver damage of streptozotocin-induced diabetic rats was investigated. It has been shown that the level of liver damage in diabetes correlates with the presence of extracellular HMGB1. In diabetic liver, this protein is structurally modified by acetylation, phosphorylation, and O-GlcNAc glycosylation, which correlates with its translocation from the nucleus to the cytoplasm and an increase in its presence in the liver and serum. Reduction of the level of extracellular HMGB1 by melatonin or ethyl pyruvate treatment of diabetic rats, shows that HMGB1 contributes to diabetic liver damage by maintaining a chronic inflammation, by lowering antioxidant defense and by reducing regeneration. Extracellular HMGB1 activates MAPK/NF-κB p65 and JAK1/STAT3 signaling pathways through interactions with the TLR4 receptor, thus contributing increased production of proinflammatory cytokines TNF-α and IL-6 and the acute-phase protein, haptoglobin. By stimulating the NF-κB p65 inflammatory pathway, HMGB1 acts negatively on the cytoprotective response of the diabetic liver, by disabling Nrf2 protein activity, which is responsible for reduction of inflammation and antioxidant enzymes production. Activated HMGB1/TLR4 axis reduces regenerative potential of the liver by increasing the presence of negative cell cycle regulators - proteins p53 and p21, and also by decreasing the level of cyclin D1. The obtained results indicate the complexity of HMGB1 protein action in diabetes and underlines the importance of preventing the release of HMGB1 or blockage of HMGB1/TLR4 axis in order to delay the occurrence of liver damage

Structural and functional characterization of HMGB1 protein in rat liver during experimentally-induced diabetes type 1

Оксидативни стрес и хронична инфламација сматрају се главним узроцима појаве дијабетичних компликација, међу којима су и оштећења јетре. Важну улогу медијатора ових процеса може имати ендогени протеин HMGB1, који у ванћелијску средину доспева из некротичних, оштећених и активираних ћелија. Како је у дијабетесу понашање HMGB1 протеина слабо изучавано, у овој докторској дисертацији испитиван је допринос HMGB1 оштећењима јетре пацова са стрептозотоцином-изазваним ДТ1. Показано је да ниво оштећења јетре током дијабетесa корелише са присуством ванћелијског HMGB1. Овај протеин, у дијабетичној јетри, бива структурно модификован ацетилацијом, фосфорилацијом и O-GlcNAc гликозилацијом што корелише са његовим изласком из једра ћелија у цитоплазму и повећањем његовог присуства у јетри и серуму. Резултати у вези са снижавањeм нивоа ванћелијског HMGB1 третманом дијабетичних пацова мелатонином или етил пируватом, указују да HMGB1 доприноси оштећењу јетре у дијабетесу одржавањем стања хроничне инфламације, стишавањем антиоксидативне одбране и стишавањем регенерације. Ванћелијски HMGB1 кроз интеракције са TLR4 рецептором активира MAPК/NF-κB p65 и ЈАК1/STAT3 сигналне путеве, доприносећи повећању продукције проинфламацијских цитокина TNF-α и IL-6 и акутно-фазног протеина хаптоглобина. Подстицањем NF-κB p65 инфламацијског пута, HMGB1 делује негативно на цитопротективни одговор у дијабетичној јетри тако што онемогућава активност Nrf2 протеина, одговорног за стишавање инфламације и продукцију антиоксидативних ензима. На стишавање регенеративног потенцијала јетре, активирана HMGB1/TLR4 оса утиче преко увећања присуства негативних регулатора ћелијског циклуса - протеина p53 и p21, и смањењем нивоа циклина D1. Добијени резултати указују на сложеност деловања HMGB1 протеина у дијабетесу и на значај спречавања ослобађања HMGB1 или блокаде HMGB1/TLR4 осе у циљу одлагања настанка оштећења јетре.Oxidative stress and chronic inflammation are considered to be the main causes of diabetic complications, one of which is liver damage. An important mediator of these processes may be the endogenous HMGB1 protein, when released into the extracellular environment from the necrotic, damaged or activated cells. As the HMGB1 role in diabetes was insufficiently studied, in this doctoral dissertation the contribution of HMGB1 to liver damage of streptozotocin-induced diabetic rats was investigated. It has been shown that the level of liver damage in diabetes correlates with the presence of extracellular HMGB1. In diabetic liver, this protein is structurally modified by acetylation, phosphorylation, and O-GlcNAc glycosylation, which correlates with its translocation from the nucleus to the cytoplasm and an increase in its presence in the liver and serum. Reduction of the level of extracellular HMGB1 by melatonin or ethyl pyruvate treatment of diabetic rats, shows that HMGB1 contributes to diabetic liver damage by maintaining a chronic inflammation, by lowering antioxidant defense and by reducing regeneration. Extracellular HMGB1 activates MAPK/NF-κB p65 and JAK1/STAT3 signaling pathways through interactions with the TLR4 receptor, thus contributing increased production of proinflammatory cytokines TNF-α and IL-6 and the acute-phase protein, haptoglobin. By stimulating the NF-κB p65 inflammatory pathway, HMGB1 acts negatively on the cytoprotective response of the diabetic liver, by disabling Nrf2 protein activity, which is responsible for reduction of inflammation and antioxidant enzymes production. Activated HMGB1/TLR4 axis reduces regenerative potential of the liver by increasing the presence of negative cell cycle regulators - proteins p53 and p21, and also by decreasing the level of cyclin D1. The obtained results indicate the complexity of HMGB1 protein action in diabetes and underlines the importance of preventing the release of HMGB1 or blockage of HMGB1/TLR4 axis in order to delay the occurrence of liver damage

Anti-melanoma effects of ingenanes isolated from Euphorbia species

In this research, from two species, E. palustris and E. lucida, four ingenane derivatives were isolated. Their anticancer effects were evaluated in the human melanoma – 518A2 cell line and compared with the effects of ingenolmebutate. Selectivity towards human melanoma cells was determined using normal human keratinocytes – HaCaT.11th Conference on Medicinal and Aromatic Plants of Southeast European Countries, (11th CMAPSEEC), Ohrid, North Macedonia, 6–10 October 202

Инхибиција аутофагије сензитизује ћелије глиобластома на инхибиторе Срц тирозин-киназе, деривате пиразоло[3,4- д]пиримидина Si306 и pro-Si306

Glioblastom je jedan od najagresivnijih tumora mozga koji karakteriše infiltrirajuća priroda, intenzivna proliferacija i rezistencija na terapiju. Ćelije glioblastoma imaju visoku ekspresiju Src tirozin-kinaze koja reguliše proliferaciju, preživljavanje i invazivnost tumorskih ćelija čineći je potencijalnom metom za terapiju. Inhibitori tirozin-kinaza mogu indukovati autofagiju koja deluje protektivno na tumorske ćelije. Sposobnost inhibitora Src tirozin-kinaze, derivata pirazolo[3,4-d]pirimidina Si306 i njegovog proleka pro-Si306, da indukuju autofagiju ispitana je na ćelijskoj liniji humanog glioblastoma U87 i njenoj varijanti sa višestrukom rezistencijom na lekove U87-TxR. Tretman ovim jedinjenjima uzrokovao je pojavu autofagozoma u ćelijama nakon 3 sata, a efekat na indukciju autofagije opstao je i nakon 48 sati što je utvrđeno analizom markera autofagije LC3 i p62. Inhibicija autofagnog fluksa bafilomicinom A1 značajno je uvećala postojeće anti-proliferativno dejstvo Si306 i pro-Si306. Takođe, kombinovani tretmani Src inhibitora sa bafilomicinom A1 doveli su do nekroze nakon 48 sati. Dobijeni rezultati sugerišu da autofagija indukovana ovim jedinjenjima ima zaštitnu ulogu u ćelijama glioblastoma i da se modulacija autofagije može koristiti za senzitizaciju ćelija glioblastoma na inhibitore Src tirozin-kinaze. Pored toga, pomenuti efekti Si306 i pro-Si306 nisu umanjeni prisustvom višestruko- rezistentnog fenotipa, što ovim jedinjenjima daje potencijal za lečenje rezistentnih tumora.Глиобластом је један од најагресивнијих тумора мозга који карактерише инфилтрирајућа природа, интензивна пролиферација и резистенција на терапију. Ћелије глиобластома имају високу експресију Срц тирозин-киназе која регулише пролиферацију, преживљавање и инвазивност туморских ћелија чинећи је потенцијалном метом за терапију. Инхибитори тирозин-киназа могу индуковати аутофагију која делује протективно на туморске ћелије. Способност инхибитора Срц тирозин-киназе, деривата пиразоло[3,4-д]пиримидина Si306 и његовог пролека pro-Si306, да индукују аутофагију испитана је на ћелијској линији хуманог глиобластома U87 и њеној варијанти са вишеструком резистенцијом на лекове U87-TxR. Третман овим једињењима узроковао је појаву аутофагозома у ћелијама након 3 сата, а ефекат на индукцију аутофагије опстао је и након 48 сати што је утврђено анализом маркера аутофагије LC3 и p62. Инхибиција аутофагног флукса бафиломицином А1 значајно је увећала постојеће анти-пролиферативно дејство Si306 и pro-Si306. Такође, комбиновани третмани Срц инхибитора са бафиломицином А1 довели су до некрозе након 48 сати. Добијени резултати сугеришу да аутофагија индукована овим једињењима има заштитну улогу у ћелијама глиобластома и да се модулација аутофагије може користити за сензитизацију ћелија глиобластома на инхибиторе Срц тирозин-киназе. Поред тога, поменути ефекти Si306 и pro-Si306 нису умањени присуством вишеструко- резистентног фенотипа, што овим једињењима даје потенцијал за лечење резистентних тумора.Knjiga sažetaka: Treći Kongres biologa Srbije, Zlatibor, Srbija 21 - 25. 9. 2022

Autophagy Inhibition Enhances Anti-Glioblastoma Effects of Pyrazolo[3,4-d]pyrimidine Tyrosine Kinase Inhibitors

Drug resistance presents a major obstacle to the successful treatment of glioblastoma. Autophagy plays a key role in drug resistance, particularly in relation to targeted therapy, which has prompted the use of autophagy inhibitors to increase the effectiveness of targeted therapeutics. The ability of two Src tyrosine kinase inhibitors, Si306 and its prodrug pro-Si306, to induce autophagy was evaluated in the human glioblastoma cell line U87 and its multidrug-resistant counterpart U87-TxR. Autophagy markers were assessed by flow cytometry, microscopy, and Western blot, and induction of autophagy by these compounds was demonstrated after 3 h as well as 48 h. The effects of Si306 and pro-Si306 on cell proliferation and cell death were examined in the presence or absence of autophagy inhibition by bafilomycin A1. Combined treatments of Si306 and pro-Si306 with bafilomycin A1 were synergistic in nature, and the inhibition of autophagy sensitized glioblastoma cells to Src tyrosine kinase inhibitors. Si306 and pro-Si306 more strongly inhibited cell proliferation and triggered necrosis in combination with bafilomycin A1. Our findings suggest that modulation of Si306- and pro-Si306-induced autophagy can be used to enhance the anticancer effects of these Src tyrosine kinase inhibitors and overcome the drug-resistant phenotype in glioblastoma cells

Anti-invasive effects of CXCR4 and FAK inhibitors in non-small cell lung carcinomas with mutually inactivated p53 and PTEN tumor suppressors

Non-small cell lung carcinoma (NSCLC) is the most common type of lung cancer. At the time of diagnosis, a large percentage of NSCLC patients have already developed metastasis, responsible for extremely high mortality rates. CXCR4 receptor and focal adhesion kinase (FAK) are known to regulate such invasive cancer behavior. Their expression is downregulated by p53 and PTEN tumor suppressors which are commonly co-inactivated in NSCLC patients and contribute to metastasis. Therefore, targeting CXCR4 or FAK seems to be a promising strategy in suppressing metastatic spread of p53/PTEN deficient NSCLCs. In this study, we first examined the invasive characteristics of NSCLC cells with suppressed p53 and PTEN activity using wound healing, gelatin degradation and invasion assays. Further, changes in the expression of CXCR4 and FAK were evaluated by RT-qPCR and Western Blot analysis. Finally, we tested the ability of CXCR4 and FAK inhibitors (WZ811 and PF-573228, respectively) to suppress the migratory and invasive potential of p53/PTEN deficient NSCLC cells, in vitro and in vivo using metastatic models of human NSCLC. Our results showed that cells with mutually inactive p53 and PTEN have significantly increased invasive potential associated with hyperactivation of CXCR4 and FAK signaling pathways. Treatments with WZ811 and PF-573228 inhibitors significantly reduced migratory and invasive capacity in vitro and showed a trend of improved survival in vivo. Accordingly, we demonstrated that p53/PTEN deficient NSCLCs have extremely invasive phenotype and provided a rationale for the use of CXCR4 or FAK inhibitors for the suppression of NSCLC dissemination.This is a post-peer-review, pre-copyedit version of an article published in Investigational New Drugs. The final authenticated version is available online at: [http://dx.doi.org/10.1007/s10637-017-0494-4

Modulation of diabetes-related liver injury by the HMGB1/TLR4 inflammatory pathway.

Chronic inflammation plays an essential role in the development of diabetic complications. Understanding the molecular mechanisms that support inflammation is a prerequisite for the design of novel anti-inflammatory therapies. These would take into consideration circulating levels of cytokines and damage-associated molecular patterns (DAMPs) that include the high mobility group box 1 (HMGB1) protein which, in part, promotes the inflammatory response through TLR4 signaling. The liver, as the source of circulating cytokines and acute-phase proteins, contributes to the control of systemic inflammation. We previously found that liver injury in streptozotocin-induced diabetic rats correlated with the level of oxidative stress, increased expression of HMGB1, and with the activation of TLR4-mediated cell death pathways. In the present work, we examined the effects of ethyl pyruvate (EP), an inhibitor of HMGB1 release/expression, on the modulation of activation of the HMGB1/TLR4 inflammatory cascade in diabetic liver. We observed that increased expression of inflammatory markers, TNF-α, IL-6, and haptoglobin in diabetic liver was associated with increased HMGB1/TLR4 interaction, activation of MAPK (p38, ERK, JNK)/NF-κB p65 and JAK1/STAT3 signaling pathways, and with decreased expression of Nrf2-regulated antioxidative enzymes. The reduction in HMGB1 expression as the result of EP administration reduced the pro-inflammatory activity of HMGB1 and exerted a protective effect on diabetic liver, which was observed as improved liver histology and antioxidant and inflammatory statuses. Our results suggest that prevention of HMGB1 release and blockage of the HMGB/TLR4 axis represents a potentially effective therapeutic strategy aimed at ameliorating diabetes-induced inflammation and ensuing liver injury.This is a post-peer-review, pre-copyedit version of an article published in Journal of Physiology and Biochemistry. The final authenticated version is available online at: [http://dx.doi.org/10.1007/s13105-018-0626-0

New anti-glioblastoma strategy with natural compounds sclareol and doxorubicin

Background: Doxorubicin (DOX) has been very effective against glioblastoma invitro. Its application in vivo is hampered because it cannot pass the blood–brainbarrier (BBB). Significant research efforts are invested to overcome this limitation.Sclareol (SC) is an aromatic compound naturally found in clary sage. Thecombination of SC and DOX showed promising effects in different tumor types invitro and in vivo. Therefore, we tested their combination and innovative hybridmolecules (SC:DOX) on glioblastoma cells with the expression of P-glycoprotein, amajor component of BBB and cancer multidrug resistance marker. Methods:Cytotoxicity and selectivity towards glioblastoma cells of SC, DOX, theircombination, and SC:DOX were examined by MTT assay. The effect of SC on DOXaccumulation was determined by flow cytometry. We also studied SC:DOXaccumulation, cellular uptake, localization imaging, and DNA damage induction.Results: The effects of simultaneous SC and DOX treatments demonstrated theconsiderable potential of SC to reverse DOX resistance in glioblastoma cells andincrease DOX accumulation. SC:DOX hybrids, named CON1 and CON2 were lesscytotoxic than DOX, but with reduced resistance and increased selectivity towardsglioblastoma cells. Cellular uptake of CON1 and CON2 was increased in glioblastomacells compared to DOX. Perinuclear localization of CON1 and CON2 vs. nuclearlocalization of DOX as well as no DNA damaging effects suggest a differentmechanism of action for SC:DOX. Conclusion: The combination of SC and DOX, andtheir innovative hybrids, could be considered a promising strategy that can overcomethe limitations of DOX application in glioblastoma.Kanazir S, Savić D, editors. Book of abstracts: 8th Congress of Serbian neuroscience society with international participation; 2023 May 31 - Jun 2; Belgrade, Serbia. Belgrade : Serbian Neuroscience Society; 2023. p. 71

Alpha-1 antitrypsin expression is upregulated in multidrug-resistant cancer cells

Identification of the signature molecular profiles involved in therapy resistance is of vital importance in developing new strategies for treatments and disease monitoring. Protein alpha-1 antitrypsin (AAT, encoded by SERPINA1 gene) is an acute-phase protein, and its high expression has been linked with unfavorable clinical outcome in different types of cancer; however, data on its involvement in therapy resistance are still insufficient. We analyzed SERPINA1 mRNA expression in three different multidrug-resistant (MDR) cell lines—U87-TxR, NCI-H460/R, and DLD1-TxR—and in U87 cells grown in alginate microfibers as a 3D cellular model of glioblastoma. Expression of IL-6 as a major modulator of SERPINA1 was also analyzed. Additionally, AAT protein expression in MDR cells was analyzed by immunofluorescence. SERPINA1 gene expression and AAT protein expression were significantly upregulated in all the tested MDR cell lines compared with their sensitive counterparts. Moreover, SERPINA1 was significantly upregulated in 3D models of glioblastoma, previously found to have upregulated drug-resistance-related gene expression compared with 2D cells. With the exception of NCI-H460/R, in all cell lines as well as in a 3D model of U87 cells, increase in SERPINA1 expression correlated with the increase in IL-6 expression. Our results indicate that AAT could be utilized as a biomarker of therapy resistance in cancer; however, further studies are needed to elucidate the mechanisms driving AAT upregulation in therapy resistance and its biological significance in this process

Oxidative stress-dependent contribution of HMGB1 to the interplay between apoptosis and autophagy in diabetic rat liver.

The progression of oxidative stress, resulting cell damage, and cell death underlies the etiology of liver damage/dysfunction as a complication of diabetes. High-mobility group box 1 (HMGB1) protein, a chromatin-binding nuclear protein and damage-associated molecular pattern molecule, is integral to oxidative stress and signaling pathways regulating cell death and cell survival. We previously found that in streptozotocin (STZ)-induced diabetic rats, reduction of oxidative stress after melatonin administration lowered necrotic cell death and increased expression of HMGB1 and hepatocellular damage. In the present study, we examined whether alleviation of diabetes-attendant oxidative stress and ensuing change in HMGB1 expression influence the dynamic equilibrium between apoptosis/autophagy and liver damage. We observed that elevated HMGB1 protein levels in diabetic rat liver accompanied increased interactions of HMGB1 with TLR4 and RAGE, and activation of the intrinsic apoptotic pathway and Beclin 1-dependent autophagy. The absence of p62 degradation in diabetic rat liver pointed to defective autophagy which was responsible for lower autophagosome/autophagolysosome formation and an increased apoptosis/autophagy ratio. Compared to diabetic rats, in melatonin-treated diabetic rats, the structure of liver cells was preserved, HMGB1/TLR4 interaction and downstream apoptotic signaling were significantly reduced, HMGB1/Beclin 1 colocalization and interactions were augmented and Beclin 1-mediated autophagy, mithophagy in particular, were increased. We concluded that in mild oxidative stress, HMGB1 is cytoprotective, whereas in intense oxidative stress, HMGB1 actions promote cell death and liver damage. Since reduced HMGB1 binds to RAGE but not to TLR4, redox modification of HMGB1 as a mechanism regulating the cross-talk between apoptosis and autophagy in diabetes is discussed.This is a post-peer-review, pre-copyedit version of an article published in Journal of Physiology and Biochemistry. The final authenticated version is available online at: [http://dx.doi.org/10.1007/s13105-017-0574-0