The role of autophagy in the in vitro antileukemic effect of cytarabine and idarubicin

Autofagija, proces programirane ćelijske razgradnje unutarćelijskog sadržaja, je uključena u regulaciju preživljavanja i smrti ćelija kancera. U ovoj doktorskoj disertaciji je po prvi put ispitivana sposobnost antileukemijskih lekova citarabina i idarubicina da indukuju autofagiju u različitim humanim leukemijskim ćelijskim linijama i mononuklearnim ćelijama periferne krvi (MNPK) pacijenata obolelih od leukemije in vitro. Takođe, ispitivani su unutarćelijski mehanizmi odgovorni za indukciju autofagije, kao i uloga autofagije u citotoksičnosti ovih lekova. Vijabilitet REH, HL-60, K562 leukemijskih ćelijskih linija, MNPK pacijenata obolelih od leukemije i MNPK zdravih kontrola je određivan merenjem aktivnosti kisele fosfataze i mitohondrijalnih dehidrogenaza. Protočna citofluorimetrija je korišćena za detekciju apoptoze i zakišeljavanja citoplazme. Indukcija autofagije je ispitivana fluorescentnom mikroskopijom (detekcija unutarćelijskih kiselih vezikula obojenih akridin oranžom), transmisionom elektronskom mikroskopijom (posmatranje autofagnih vezikula) i imunoblot analizom (konverzija LC3-I u LC3-II, degradacija SQSTM1/p62). Aktivacija signalnih puteva koji učestvuju u regulaciji autofagije je analizirana imunoblot metodom. Uloga autofagije u citotoksičnosti citarabina i idarubicina je ispitivana primenom farmakološke inhibicije bafilomicinom A1, hlorokinom, vortmaninom i amonijum hloridom, kao i genetske inaktivacije ekspresije beklina-1, LC3β i SQSTM1 transfekcijom odgovarajućim malim interferirajućim RNK. Citarabin i idarubicin su izazvali povećanje unutarćelijske kiselosti i pojavu autofagnih vezikula sa delimično razgrađenim ćelijskim sadržajem u leukemijskim ćelijskim linijama. Antileukemijski lekovi su stimulisali razgradnju supstrata autofagije SQSTM1 i povećali konverziju LC3-I u LC3-II formu asociranu autofagozomima u odsustvu ili prisustvu inhibitora proteolize, ukazujući tako na povećanje autofagnog fluksa. Oba leka su smanjila fosforilaciju mTOR kinaze, glavnog negativnog regulatora autofagije i njegovog supstrata p70S6 kinaze, dok je tretman mTOR aktivatorom leucinom sprečio indukciju autofagije. Idarubicin je suprimirao aktivnost mTOR aktivatora...Autophagy, a process of programmed cellular self-digestion, has been implicated in regulation of cancer cell survival and death. The present study investigated for the first time the ability of antileukemic drugs cytarabine and idarubicin to induce autophagy in different human leukemic cell lines and peripheral blood mononuclear cells (PBMC) from leukemia patients in vitro. Intracellular mechanisms responsible for the induction of autophagy, as well as the role of autophagy in cytotoxicity of these drugs were also investigated. Cell viability of REH, HL-60, K562 leukemic cell lines, and PBMC from leukemic patients and healthy controls was determined by measuring the acid phosphatase and mitochondrial succinate dehydrogenase activity. Flow cytometry was used for the detection of apoptosis and intracellular acidification. Autophagy induction was assessed by fluorescent microscopy (detection of acridine orange stained intracellular acidic vesicles), by transmission electron microscopy (observation of autophagic vacuoles), as well as by immunoblot analysis of LC3 conversion and SQSTM1/p62 proteolysis. Activation of autophagy-regulating signaling pathways was analyzed by immunoblotting. Pharmacological inhibition of autophagy with bafilomycin A1, chloroquine, wortmannin, and NH4Cl or RNA interference-mediated knockdown of beclin-1, LC3β and SQSTM1 were used to determine the role of the autophagy in cytotoxicity of antileukemic drugs. Cytarabine and idarubicin induced an increase in intracellular acidification and appearance of autophagic vesicles with partially digested cellular components in leukemic cell lines. Antileukemic drugs stimulated the degradation of autophagic target SQSTM1 and enhanced the conversion of LC3-I to autophagosome-associated LC3-II in the absence or presence of proteolysis inhibitors, thus indicating the increase in autophagic flux..

Graphene quantum dots protect SH-SY5Y neuronal cells from SNP-induced apoptotic death

Introduction: We examined the molecular mechanisms of graphene quantum dot (GQD)- mediated protection of SH-SY5Y human neuroblastoma cells from oxidative/nitrosative stress induced by iron-nitrosyl complex sodium nitroprusside (SNP). Methods: GQD was produced by electrochemical oxidation of graphite and characterized by AFM, UVVIS and FTIR spectroscopy. The antioxidant activity of GQD in cell-free conditions was assessed by DPPH, NBT and EPR analysis. The neuroprotective potential of GQD was determined by cell viability assays MTT, CV. Flow cytometry was used to assess markers of apoptosis and GQD scavenging of intracellular ROS/RNS as well. Cellular internalization of GQD was determined using TEM. Results: GQD prevented SNP-induced apoptosis, caspase activation and mitochondrial depolarization in neuroblastoma cells. Although GQD diminished the NO levels in SNP-treated cells, NO scavengers displayed only a slight protection. GQD significantly protected SH-SY5Y cells from neurotoxicity of lightexhausted SNP, incapable of producing NO, implying that protective mechanism is independent of NO-scavenging. GQD reduced SNP-triggered increase in intracellular levels of ROS, particularly •OH, O2•− in cells and cell-free condition. Nonselective antioxidants, •OH scavengers and iron chelators, mimicked GQD cytoprotection, indicating that GQD protect cells by neutralizing •OH generated in the Fenton reaction. Cellular GQD internalization was required for optimal protection since the removal of extracellular GQD by extensive washing partly diminished their protective effect, suggesting that GQD exerted neuroprotective effect intra- and extracellularly. Conclusion: By demonstrating that GQD protect neuroblastoma cells from SNP-induced apoptosis by •OH/NO scavenging, our results suggest that GQD could be valuable candidates for treatment of neurodegenerative diseases associated with oxidative/nitrosative stress

Autophagy receptor p62 regulates SARS-CoV-2-induced inflammation in COVID-19

Introduction: Since the interaction between autophagy and virus-induced inflammation is complex, we investigated the interplay between autophagy and inflammation in COVID-19 patients and THP-1 cells expressing SARS-Cov2 proteins NSP5 and ORF3a. Methods: Autophagy markers in blood from 19 control subjects and 26 COVID-19 patients at hospital admission and one week later were measured by ELISA, while cytokine levels were examined by flow cytometric bead immunoassay. The level of p62 in cells and its concentration in cell culture supernatants was measured by immunoblot/ELISA. The mRNA levels of proinflammatory cytokines were measured by RT-qPCR. Results: IFN-α, TNF, IL-6, IL-8, IL-17, IL-33, and IFN-γ were elevated in COVID-19 patients at both time points, whereas IL-10 and IL-1β were elevated at admission and one week later, respectively. Autophagy markers LC3 and ATG5 were unchanged in COVID-19. The concentration of autophagic cargo receptor p62 was significantly lower and positively correlated with TNF, IL-10, IL-17, and IL-33 at hospital admission,returning to normal levels after one week. The expression of SARS-CoV-2 proteins NSP5 or ORF3a in THP-1 cells caused an autophagy-independent decrease/autophagy-inhibition-dependent increase of intracellular and secreted p62. This was associated with an NSP5-mediated decrease in TNF/IL-10 mRNA and an ORF3a-mediated increase in TNF/IL-1β/IL-6/IL-10/IL-33 mRNA levels. A genetic knockdown of p62 mimicked the immunosuppressive effect of NSP5, while a p62 increase in autophagy-deficient cells mirrored the immunostimulatory action of ORF3a. Conclusion: The autophagy receptor p62 is reduced in acute COVID-19, and the balance between autophagy-independent decrease and autophagy blockade-dependent increase of p62 levels could affect SARS-CoV-induced inflammation

The role of autophagy in the in vitro antileukemic effect of cytarabine and idarubicin

Autofagija, proces programirane ćelijske razgradnje unutarćelijskog sadržaja, je uključena u regulaciju preživljavanja i smrti ćelija kancera. U ovoj doktorskoj disertaciji je po prvi put ispitivana sposobnost antileukemijskih lekova citarabina i idarubicina da indukuju autofagiju u različitim humanim leukemijskim ćelijskim linijama i mononuklearnim ćelijama periferne krvi (MNPK) pacijenata obolelih od leukemije in vitro. Takođe, ispitivani su unutarćelijski mehanizmi odgovorni za indukciju autofagije, kao i uloga autofagije u citotoksičnosti ovih lekova. Vijabilitet REH, HL-60, K562 leukemijskih ćelijskih linija, MNPK pacijenata obolelih od leukemije i MNPK zdravih kontrola je određivan merenjem aktivnosti kisele fosfataze i mitohondrijalnih dehidrogenaza. Protočna citofluorimetrija je korišćena za detekciju apoptoze i zakišeljavanja citoplazme. Indukcija autofagije je ispitivana fluorescentnom mikroskopijom (detekcija unutarćelijskih kiselih vezikula obojenih akridin oranžom), transmisionom elektronskom mikroskopijom (posmatranje autofagnih vezikula) i imunoblot analizom (konverzija LC3-I u LC3-II, degradacija SQSTM1/p62). Aktivacija signalnih puteva koji učestvuju u regulaciji autofagije je analizirana imunoblot metodom. Uloga autofagije u citotoksičnosti citarabina i idarubicina je ispitivana primenom farmakološke inhibicije bafilomicinom A1, hlorokinom, vortmaninom i amonijum hloridom, kao i genetske inaktivacije ekspresije beklina-1, LC3β i SQSTM1 transfekcijom odgovarajućim malim interferirajućim RNK. Citarabin i idarubicin su izazvali povećanje unutarćelijske kiselosti i pojavu autofagnih vezikula sa delimično razgrađenim ćelijskim sadržajem u leukemijskim ćelijskim linijama. Antileukemijski lekovi su stimulisali razgradnju supstrata autofagije SQSTM1 i povećali konverziju LC3-I u LC3-II formu asociranu autofagozomima u odsustvu ili prisustvu inhibitora proteolize, ukazujući tako na povećanje autofagnog fluksa. Oba leka su smanjila fosforilaciju mTOR kinaze, glavnog negativnog regulatora autofagije i njegovog supstrata p70S6 kinaze, dok je tretman mTOR aktivatorom leucinom sprečio indukciju autofagije. Idarubicin je suprimirao aktivnost mTOR aktivatora...Autophagy, a process of programmed cellular self-digestion, has been implicated in regulation of cancer cell survival and death. The present study investigated for the first time the ability of antileukemic drugs cytarabine and idarubicin to induce autophagy in different human leukemic cell lines and peripheral blood mononuclear cells (PBMC) from leukemia patients in vitro. Intracellular mechanisms responsible for the induction of autophagy, as well as the role of autophagy in cytotoxicity of these drugs were also investigated. Cell viability of REH, HL-60, K562 leukemic cell lines, and PBMC from leukemic patients and healthy controls was determined by measuring the acid phosphatase and mitochondrial succinate dehydrogenase activity. Flow cytometry was used for the detection of apoptosis and intracellular acidification. Autophagy induction was assessed by fluorescent microscopy (detection of acridine orange stained intracellular acidic vesicles), by transmission electron microscopy (observation of autophagic vacuoles), as well as by immunoblot analysis of LC3 conversion and SQSTM1/p62 proteolysis. Activation of autophagy-regulating signaling pathways was analyzed by immunoblotting. Pharmacological inhibition of autophagy with bafilomycin A1, chloroquine, wortmannin, and NH4Cl or RNA interference-mediated knockdown of beclin-1, LC3β and SQSTM1 were used to determine the role of the autophagy in cytotoxicity of antileukemic drugs. Cytarabine and idarubicin induced an increase in intracellular acidification and appearance of autophagic vesicles with partially digested cellular components in leukemic cell lines. Antileukemic drugs stimulated the degradation of autophagic target SQSTM1 and enhanced the conversion of LC3-I to autophagosome-associated LC3-II in the absence or presence of proteolysis inhibitors, thus indicating the increase in autophagic flux..

Intraoperative electrochemotherapy of the posterior resection surface after pancreaticoduodenectomy

Background: Despite extensive research in recent decades, pancreatic cancer continues to be among the most lethal forms of cancer, with no substantial increase in survival rates. Local recurrences account for approximately 30 per cent of all disease recurrences. With the intent to improve survival, we designed a novel, hybrid treatment strategy consisting of surgical resection and additional intraoperative electrochemotherapy of the posterior resection surface. We present the study protocols and preliminary findings of a prospective pilot study investigating this treatment approach. Methods: Consenting patients with resectable pancreatic head ductal adenocarcinoma who met the inclusion criteria were enrolled in the study. After surgical resection, electrochemotherapy with bleomycin was performed using plate electrodes to cover the area between anatomical landmarks. Results: Electrochemotherapy of the posterior resection surface was feasible in all 7 patients. We observed pancreatic fistula grade B in only one patientall other noted complications were Clavien-Dindo grade 2 or less. The hospital mortality was 0%. Conclusions: Our preliminary results suggest that a hybrid approach combining surgery with intraoperative electrochemotherapy is safe and feasible

A prospective phase II study evaluating intraoperative electrochemotherapy of hepatocellular carcinoma

The aim of this clinical study was to investigate the effectiveness and long-term safety of electrochemotherapy as an emerging treatment for HCC in patients not suitable for other treatment options. A prospective phase II clinical study was conducted in patients with primary HCC who were not suitable for other treatment options according to the Barcelona Clinic Liver Cancer classification. A total of 24 patients with 32 tumors were treated by electrochemotherapy. The procedure was effective, feasible, and safe with some procedure-related side effects. The responses of the 32 treated nodules were: 84.4% complete response (CR), 12.5% partial response (PR), and 3.1% stable disease (SD). The treatment was equally effective for nodules located centrally and peripherally. Electrochemotherapy provided a durable response with local tumor control over 50 months of observation in 78.0% of nodules. The patient responses were: 79.2% CR and 16.6% PR. The median progression-free survival was 12 months (range 2.7–50), and the overall survival over 5 years of observation was 72.0%. This prospective phase II clinical study showed that electrochemotherapy was an effective, feasible, and safe option for treating HCC in patients not suitable for other treatment options

In vitro antiglioma action of indomethacin is mediated via AMP-activated protein kinase/mTOR complex 1 signalling pathway

We investigated the role of the intracellular energy-sensing AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in the in vitro antiglioma effect of the cyclooxygenase (COX) inhibitor indomethacin. Indomethacin was more potent than COX inhibitors diclofenac, naproxen, and ketoprofen in reducing the viability of U251 human glioma cells. Antiglioma effect of the drug was associated with p21 increase and G2M cell cycle arrest, as well as with oxidative stress, mitochondrial depolarization, caspase activation, and the induction of apoptosis. Indomethacin increased the phosphorylation of AMPK and its targets Raptor and acetyl-CoA carboxylase (ACC), and reduced the phosphorylation of mTOR and mTOR complex 1 (mTORC1) substrates p70S6 kinase and PRAS40 (Ser183). AMPK knockdown by RNA interference, as well as the treatment with the mTORC1 activator leucine, prevented indomethacin-mediated mTORC1 inhibition and cytotoxic action, while AMPK activators metformin and AICAR mimicked the effects of the drug. AMPK activation by indomethacin correlated with intracellular ATP depletion and increase in AMP/ATP ratio, and was apparently independent of COX inhibition or the increase in intracellular calcium. Finally, the toxicity of indomethacin towards primary human glioma cells was associated with the activation of AMPK/Raptor/ACC and subsequent suppression of mTORC1/S6K. By demonstrating the involvement of AMPK/mTORC1 pathway in the antiglioma action of indomethacin, our results support its further exploration in glioma therapy.The International Journal of Biochemistry & Cell Biology (2017), 83: 84-9

Safety and feasibility of electrochemotherapy of the pancreas in a porcine model

Objectives: The use of thermal ablative therapies in the pancreatic tumors is limited because of the risk of the vessel injury and potential pancreatitis or fistula formation. Electrochemotherapy (ECT) is an ablative therapy with established role in the treatment of cutaneous and liver tumors. This study was designed to evaluate the safety and feasibility of ECT of the pancreas in a porcine survival model. Methods: In the first group, 4 animals underwent computed tomography (CT)%guided percutaneous ECT with bleomycin of the pancreatic tail. In the second group (4 animals), the intraoperative ECTwith bleomycin of pancreatic tail and head was performed. Animals were followed for 7 days and then killed. Clinical parameters, CT imaging, laboratory, and histologic analysis were performed. Results: All pigs survived the ECT procedure and none of them developed clinical signs of acute pancreatitis or related complications. There were no signs of acute pancreatitis or damage to the large vessels present in the follow-up CT scans. No significant change in laboratory parameters was obtained after procedure. Conclusions: This study shows that ECT with bleomycin is feasible and safe in the pancreatic parenchyma. Clinical studies are needed to evaluate the efficacy of ECT in pancreatic cancer

Graphene quantum dot antioxidant and proautophagic actions protect SH-SY5Y neuroblastoma cells from oxidative stress-mediated apoptotic death

We investigated the ability of graphene quantum dot (GQD) nanoparticles to protect SH-SY5Y human neuro blastoma cells from oxidative/nitrosative stress induced by iron-nitrosyl complex sodium nitroprusside (SNP). GQD reduced SNP cytotoxicity by preventing mitochondrial depolarization, caspase-2 activation, and subsequent apoptotic death. Although GQD diminished the levels of nitric oxide (NO) in SNP-exposed cells, NO scavengers displayed only a slight protective effect, suggesting that NO quenching was not the main protective mechanism of GQD. GQD also reduced SNP-triggered increase in the intracellular levels of hydroxyl radical ( • OH), superoxide anion (O2 •− ), and lipid peroxidation. Nonselective antioxidants, • OH scavenging, and iron chelators, but not superoxide dismutase, mimicked GQD cytoprotective activity, indicating that GQD protect cells by neutralizing • OH generated in the presence of SNP-released iron. Cellular internalization of GQD was required for optimal protection, since a removal of extracellular GQD by extensive washing only partly diminished their protective effect. Moreover, GQD cooperated with SNP to induce autophagy, as confirmed by the inhibition of autophagy limiting Akt/PRAS40/mTOR signaling and increase in autophagy gene transcription, protein levels of proauto phagic beclin-1 and LC3-II, formation of autophagic vesicles, and degradation of autophagic target p62. The antioxidant activity of GQD was not involved in autophagy induction, as antioxidants N-acetylcysteine and dimethyl sulfoxide failed to stimulate autophagy in SNP-exposed cells. Pharmacological inhibitors of early (wortmannin, 3-methyladenine) or late stages of autophagy (NH4Cl) efficiently reduced the protective effect of GQD. Therefore, the ability of GQD to prevent the in vitro neurotoxicity of SNP depends on both • OH/NO scavenging and induction of cytoprotective autophagy