Growth inhibition and apoptosis induced by 2 phenoxymethyl-3H-quinazolin-4-one in HL-60 leukemia cells

Aim: The aim of the study was to investigate anticancer activity of newly synthesized 2-phenoxymethyl-3H-quinazolin-4-one (PMQ). Materials and Methods: Anticancer activity of PMQ was studied towards human HL-60 leukemia cells. Antiproliferative activity of PMQ was determined by direct counting of cells using trypan blue staining technique. Apoptosis and cell cycle profile changes were analysed using internucleosomal DNA fragmentation assay and flow cytometry. Activation of caspases and changes in glutathione level were monitored using colorimetric or luminiscent methods. Results: PMQ induced concentration-dependent cytotoxicity in leukemia cells, with IC50 of 10.8 ± 0.9 µM. DNA flow cytometry analysis and DNA ladder formation assay indicated that PMQ actively induced apoptosis of cells accompanied by a block of cells in G2/M phase and a marked loss of cells in G0/G1 and S phases. Additionally, the activities of caspase-3 and caspase-9 were increased significantly and a markedly increased level of oxidized glutahione was observed. Inhibition of glutahione synthesis using buthionine sulfoximine sensitized leukemia cells to PMQ, confirming the involvement of ROS in PMQ-induced apoptosis. Conclusion: The results of this study clearly demonstrate that PMQ is a promising anticancer drug showing cytostatic and apoptotic effects toward HL-60 leukemia cells mainly through mitochondrial/caspase-9 dependent pathway.Цель: изучить антипролиферативную активность нового синтезированного 2-феноксиметил-3Н-хиназолин-4-она (PMQ). Материалы и методы: антипролиферативную активность PMQ определяли по отношению к клеткам лейкоза линии HL-60 в тесте с трипановым синим при стандартном подсчете клеток. Апоптоз и клеточный цикл оценивали с помощью проточной цитометрии и анализа фрагментации внутриядерной ДНК. Активацию каспаз и изменения уровня глутатиона определяли колориметрическими или люминисцентными методами. Результаты: PMQ индуцирует дозозависимую цитотоксичность в клетках линии HL-60 (IC50 при 10,8 ± 0,9 μM). При проведении анализа ДНК с применением проточной цитометрии и определением формирования апоптической лестницы было показано, что PMQ активно индуцирует апоптоз и блокаду клеточного цикла в G2 /M фазе митоза и выраженной потерей клеток в G0 /G1 и S фазах. Кроме того, была достоверно повышена активность каспазы-3 и -9 и выраженно увеличен уровень окисленного глутатиона. Применение бутионин сульфоксимина привело к угнетению синтеза глутатиона и повышению чувствительности клеток HL-60 к PMQ, что подтверждает факт участия РФК в PMQ-индуцированном апоптозе. Выводы: PMQ проявил себя как потенциальное противоопухолевое средство против клеток лейкоза человека HL-60 с выраженным цитостатическим и проапоптическим действием

Antiproliferative activity and apoptosis induced by 6-bromo-2-(morpholin-1-yl)-4-anilinoquinazoline on cells of leukemia lines

Quinazolines are known to be multitarget agents with broad spectrum of biological activity. Aim: To investigate anticancer activity of newly prepared 6-bromo-2-(morpholin-1-yl)-4-anilinoquinazoline (BMAQ) towards L1210, HL-60 and U-937 leukemia cells. Materials and Methods: Growth inhibition of BMAQ-treated cells was determined by cell counting using trypan blue staining technique. Apoptosis and cell cycle profile changes were analysed using internucleosomal DNA fragmentation assay, fluorescence microscopy and flow cytometry. Activity of caspase-3 was determined using colorimetric method. Results: Cell proliferation assay showed that BMAQ caused significant decrease of cell number in a dose-dependent manner. BMAQ induced cell death by apoptosis, based on results from DNA fragmentation, fluorescence microscopy and caspase-3 assays. Conclusion: Presented results clearly demonstrate that BMAQ is a promising anticancer agent with significant antiproliferative and apoptotic activities towards leukemia cells in vitro.Квиназолины известны как химиопрепараты широкого спектра действия. Цель: на моделях лейкозных клеток линий L1210, HL-60 и U-937 изучить противоопухолевую активность нового препарата 6-бромо-2-(морфолин-1-ил)-4-аналиноиназолина (BMAQ). Методы: ингибирование роста клеток под действием BMAQ изучали путем подсчета количества клеток, окрашенных трипановым синим. Апоптоз и изменения профиля клеточного цикла исследовали с помощью флуоресцентной микроскопии, электрофореза ДНК и проточной цитометрии. Активность каспазы-3 определяли колориметрическим методом. Результаты: показано, что BMAQ вызывает значительное дозозависимое уменьшение количества лейкозных клеток. При этом клетки, обработанные BMAQ, погибают путем апоптоза, что дается образованием апоптотических телец, межнуклеосомной фрагментацией ДНК и активацией каспазы-3. Выводы: представленные результаты свидетельствуют о том, что BMAQ обладает антипролиферативной и проапоптотической активностью в отношении лейкозных клеток in vitro

Advanced glycoxidation and lipoxidation end products (AGEs and ALEs): an overview of their mechanisms of formation

Advanced lipoxidation end products (ALEs) and advanced glycation end products (AGEs) have a pathogenetic role in the development and progression of different oxidative-based diseases including diabetes, atherosclerosis, and neurological disorders. AGEs and ALEs represent a quite complex class of compounds that are formed by different mechanisms, by heterogeneous precursors and that can be formed either exogenously or endogenously. There is a wide interest in AGEs and ALEs involving different aspects of research which are essentially focused on set-up and application of analytical strategies (1) to identify, characterize, and quantify AGEs and ALEs in different pathophysiological conditions ; (2) to elucidate the molecular basis of their biological effects ; and (3) to discover compounds able to inhibit AGEs/ALEs damaging effects not only as biological tools aimed at validating AGEs/ALEs as drug target, but also as promising drugs. All the above-mentioned research stages require a clear picture of the chemical formation of AGEs/ALEs but this is not simple, due to the complex and heterogeneous pathways, involving different precursors and mechanisms. In view of this intricate scenario, the aim of the present review is to group the main AGEs and ALEs and to describe, for each of them, the precursors and mechanisms of formation

DNA damage by lipid peroxidation products: implications in cancer, inflammation and autoimmunity

Oxidative stress and lipid peroxidation (LPO) induced by inflammation, excess metal storage and excess caloric intake cause generalized DNA damage, producing genotoxic and mutagenic effects. The consequent deregulation of cell homeostasis is implicated in the pathogenesis of a number of malignancies and degenerative diseases. Reactive aldehydes produced by LPO, such as malondialdehyde, acrolein, crotonaldehyde and 4-hydroxy-2-nonenal, react with DNA bases, generating promutagenic exocyclic DNA adducts, which likely contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced LPO. However, reactive aldehydes, when added to tumor cells, can exert an anticancerous effect. They act, analogously to other chemotherapeutic drugs, by forming DNA adducts and, in this way, they drive the tumor cells toward apoptosis. The aldehyde-DNA adducts, which can be observed during inflammation, play an important role by inducing epigenetic changes which, in turn, can modulate the inflammatory process. The pathogenic role of the adducts formed by the products of LPO with biological macromolecules in the breaking of immunological tolerance to self antigens and in the development of autoimmunity has been supported by a wealth of evidence. The instrumental role of the adducts of reactive LPO products with self protein antigens in the sensitization of autoreactive cells to the respective unmodified proteins and in the intermolecular spreading of the autoimmune responses to aldehyde-modified and native DNA is well documented. In contrast, further investigation is required in order to establish whether the formation of adducts of LPO products with DNA might incite substantial immune responsivity and might be instrumental for the spreading of the immunological responses from aldehyde-modified DNA to native DNA and similarly modified, unmodified and/or structurally analogous self protein antigens, thus leading to autoimmunity

Biocompatibility of oxygen-plasma-treated polystyrene substrates

The biocompatibility of polystyrene (PS) samples has been improved by treatment with weakly ionized highly non-equilibrium oxygen plasma. Samples were exposed to plasma for 30 s for which they have received a dose of ions of 4.5 × 1017 m−2 and a neutral oxygen atom dose of 3 × 10−23 m−2. Both untreated and plasma-treated samples were tested for biocompatibility according to the same procedure. Proliferation of human mammary epithelial cells (HMECs) on samples revealed a dramatically improved biocompatibility of polystyrene treated by oxygen plasma. The HMECs were deposited on all samples and incubated for 1, 2 and 6 days. MTT test revealed about two times higher activity of cell enzymes after 48 h incubation. The activity for plasma-treated samples remained much higher than for untreated samples even after 6 days of incubation when the samples were already covered with a dense film of HMECs

Laser microsurgery provides evidence for merotelic kinetochore attachments in fission yeast cells lacking Pcs1 or Clr4

In order to segregate chromosomes properly, the cell must prevent merotelic kinetochore attachment, an error that occurs when a single kinetochore is attached to microtubules emanating from both spindle poles. Merotelic kinetochore orientation represents a major mechanism of aneuploidy in mitotic mammalian cells and it is the primary mechanism of chromosome instability in cancer cells. Fission yeast mutants defective in putative microtubule-site clamp Pcs1/ Mde4 or Clr4/Swi6-dependent centromeric heterochromatin display high frequencies of lagging chromosomes during anaphase. Here, we developed an assay based on laser microsurgery to show that the stretched morphology of lagging kinetochores in pcs1Δ and clr4Δ mutant cells is due to merotelic attachment. We further show that Mde4 is regulated by Cdc2 and that Cdc2 activity prevents precocious localization of Mde4 to the metaphase spindle. Finally, we show that Pcs1/Mde4 complex shares similar features with the conserved kinetochore complex Spc24/Spc25 suggesting that these two complexes may occupy a similar functional niche

Chemistry and biochemistry of lipid peroxidation products

Oxidative stress and resulting lipid peroxidation is involved in various and numerous pathological states including inflammation, atherosclerosis, neurodegenerative diseases and cancer. This review is focused on recent advances concerning the formation, metabolism and reactivity towards macromolecules of lipid peroxidation breakdown products, some of which being considered as 'second messengers' of oxidative stress. This review relates also new advances regarding apoptosis induction, survival/proliferation processes and autophagy regulated by 4-hydroxynonenal, a major product of omega-6 fatty acid peroxidation, in relationship with detoxication mechanisms. The use of these lipid peroxidation products as oxidative stress/lipid peroxidation biomarkers is also addressed

Oxidative Stress and Cancer Heterogeneity Orchestrate NRF2 Roles Relevant for Therapy Response

Oxidative stress and its end-products, such as 4-hydroxynonenal (HNE), initiate activation of the Nuclear Factor Erythroid 2-Related Factor 2 (NRF2)/KEAP1 signaling pathway that plays a crucial role in the maintenance of cellular redox homeostasis. However, an involvement of 4-HNE and NRF2 in processes associated with the initiation of cancer, its progression, and response to therapy includes numerous, highly complex events. They occur through interactions between cancer and stromal cells. These events are dependent on many cell-type specific features. They start with the extent of NRF2 binding to its cytoplasmic repressor, KEAP1, and extend to the permissiveness of chromatin for transcription of ARE-containing genes that are NRF2 targets. This review will explore epigenetic molecular mechanisms of NRF2 transcription through the specific molecular anatomy of its promoter. It will explain the role of NRF2 in cancer stem cells, with respect to cancer therapy resistance. Additionally, it also discusses NRF2 involvement at the cross-roads of communication between tumor associated inflammatory and stromal cells, which is also an important factor involved in the response to therapy