Baicalin and Baicalein Enhance Cytotoxicity, Proapoptotic Activity, and Genotoxicity of Doxorubicin and Docetaxel in MCF-7 Breast Cancer Cells.

Breast cancer is a major health concern and the leading cause of death among women worldwide. Standard treatment often involves surgery, radiotherapy, and chemotherapy, but these come with side effects and limitations. Researchers are exploring natural compounds like baicalin and baicalein, derived from the Scutellaria baicalensis plant, as potential complementary therapies. This study investigated the effects of baicalin and baicalein on the cytotoxic, proapoptotic, and genotoxic activity of doxorubicin and docetaxel, commonly used chemotherapeutic drugs for breast cancer. The analysis included breast cancer cells (MCF-7) and human endothelial cells (HUVEC-ST), to assess potential effects on healthy tissues. We have found that baicalin and baicalein demonstrated cytotoxicity towards both cell lines, with more potent effects observed in baicalein. Both flavonoids, baicalin (167 µmol/L) and baicalein (95 µmol/L), synergistically enhanced the cytotoxic, proapoptotic, and genotoxic activity of doxorubicin and docetaxel in breast cancer cells. In comparison, their effects on endothelial cells were mixed and depended on concentration and time. The results suggest that baicalin and baicalein might be promising complementary agents to improve the efficacy of doxorubicin and docetaxel anticancer activity. However, further research is needed to validate their safety and efficacy in clinical trials.

Acrolein Induces Changes in Cell Membrane and Cytosol Proteins of Erythrocytes.

High concentrations of acrolein (2-propenal) are found in polluted air and cigarette smoke, and may also be generated endogenously. Acrolein is also associated with the induction and progression of many diseases. The high reactivity of acrolein towards the thiol and amino groups of amino acids may cause damage to cell proteins. Acrolein may be responsible for the induction of oxidative stress in cells. We hypothesized that acrolein may contribute to the protein damage in erythrocytes, leading to the disruption of the structure of cell membranes. The lipid membrane fluidity, membrane cytoskeleton, and osmotic fragility were measured for erythrocytes incubated with acrolein for 24 h. The levels of thiol, amino, and carbonyl groups were determined in cell membrane and cytosol proteins. The level of non-enzymatic antioxidant potential (NEAC) and TBARS was also measured. The obtained research results showed that the exposure of erythrocytes to acrolein causes changes in the cell membrane and cytosol proteins. Acrolein stiffens the cell membrane of erythrocytes and increases their osmotic sensitivity. Moreover, it has been shown that erythrocytes treated with acrolein significantly reduce the non-enzymatic antioxidant potential of the cytosol compared to the control.

Modulation of the Human Erythrocyte Antioxidant System by the 5- and 6-Membered Heterocycle-Based Nitroxides.

Nitroxides are stable radicals consisting of a nitroxyl group, >N-O•, which carries an unpaired electron. This group is responsible for the paramagnetic and antioxidant properties of these compounds. A recent study evaluated the effects of pyrrolidine and pyrroline derivatives of nitroxides on the antioxidant system of human red blood cells (RBCs). It showed that nitroxides caused an increase in the activity of superoxide dismutase (SOD) and the level of methemoglobin (MetHb) in cells (in pyrroline derivatives) but had no effect on the activity of catalase and lactate dehydrogenase. Nitroxides also reduced the concentration of ascorbic acid (AA) in cells but did not cause any oxidation of proteins or lipids. Interestingly, nitroxides initiated an increase in thiols in the plasma membranes and hemolysate. However, the study also revealed that nitroxides may have pro-oxidant properties. The drop in the AA concentration and the increase in the MetHb level and in SOD activity may indicate the pro-oxidant properties of nitroxides in red blood cells.

Factors Influencing Venous Remodeling in the Development of Varicose Veins of the Lower Limbs.

One of the early symptoms of chronic venous disease (CVD) is varicose veins (VV) of the lower limbs. There are many etiological environmental factors influencing the development of chronic venous insufficiency (CVI), although genetic factors and family history of the disease play a key role. All these factors induce changes in the hemodynamic in the venous system of the lower limbs leading to blood stasis, hypoxia, inflammation, oxidative stress, proteolytic activity of matrix metalloproteinases (MMPs), changes in microcirculation and, consequently, the remodeling of the venous wall. The aim of this review is to present current knowledge on CVD, including the pathophysiology and mechanisms related to vein wall remodeling. Particular emphasis has been placed on describing the role of inflammation and oxidative stress and the involvement of extracellular hemoglobin as pathogenetic factors of VV. Additionally, active substances used in the treatment of VV were discussed.

The Effect of Piperidine Nitroxides on the Properties of Metalloproteins in Human Red Blood Cells.

Nitroxides are stable, low molecular-weight radicals containing a nitroxide group that has an unpaired electron. The presence of a nitroxide group determines their redox properties. The effect of the piperidine nitroxides, Tempo, Tempol, and Tempamine, on metalloproteins (hemoglobin, superoxide dismutase, catalase) and lactate dehydrogenase in red blood cells was investigated in this research. In addition, the level of lipid peroxidation and the level of protein carbonyl groups were examined as indicators of the effect of oxidative stress. Nitroxides increased superoxide dismutase activity and oxidized hemoglobin to methemoglobin, and also slightly decreased the catalase activity of red blood cells treated with nitroxides. Tempol significantly decreased lactate dehydrogenase activity. All three nitroxides had no effect on membrane lipid peroxidation and protein oxidation. Our results confirm that nitroxides have both antioxidant and prooxidative effects in human red blood cells. The piperidine nitroxides do not initiate the oxidation of proteins and lipids in the membranes of human red blood cells.

Indoxyl sulfate induces apoptosis in mononuclear blood cells via mitochondrial pathway.

The consequence of chronic kidney disease is the accumulation of metabolic products called uremic toxins in the body. Indoxyl sulfate (IS) is a toxin with a high affinity for proteins. This study focuses on the deleterious effect of IS, especially apoptosis induction, in mononuclear blood cells (MNCs). Thus, in MNCs treated with IS at three different concentrations for 24 h, the survival, mitochondrial potential, caspases activity and expression, Bcl-2 and Bax protein expression, DNA damage, and PARP degradation were estimated. The study showed a decrease in survival and mitochondrial potential of MNCs treated with IS compared to the control. IS increased the activity of caspase 2-, 3-, 9-, and the expression of caspase 3-, and 9- in MNCs but does not affect the activity of caspase 6- and 8. The treatment of MNCs with IS also increased DNA damage and degradation of PARP. Indoxyl sulfate significantly influences the expression of Bcl-2 and Bax proteins. Indoxyl sulfate induces the programmed death of MNCs through the intrinsic mitochondrial apoptotic pathway. The observed cellular changes are mostly dose-dependent.

The Effect of Diosmin, Escin, and Bromelain on Human Endothelial Cells Derived from the Umbilical Vein and the Varicose Vein-A Preliminary Study.

In this study, we investigated the properties of human varicose vein (VV) endothelial cells (HVVEC) in comparison to the human umbilical vein endothelial cells (HUVEC). The cells were treated with three bioactive compounds with proven beneficial effects in the therapy of patients with VV, diosmin, escin, and bromelain. Two concentrations of tested drugs were used (1, 10 mg/mL), which did not affect the viability of either cell type. Escin led to a slight generation of reactive oxygen species in HUVEC cells. We observed a slight release of superoxide in HVVEC cells upon treatment with diosmin and escin. Diosmin and bromelain showed a tendency to release nitric oxide in HUVEC. Using membrane fluorescent probes, we demonstrated a reduced fluidity of HVVEC, which may lead to their increased adhesion, and, consequently, a much more frequent occurrence of venous thrombosis. For the first time, we show the mechanism of action of drugs used in VV therapy on endothelial cells derived from a VV. Studies with HVVEC have shown that tested drugs may lead to a reduction in the adhesive properties of these cells, and thus to a lower risk of thrombosis.

Diosmin and Bromelain Stimulate Glutathione and Total Thiols Production in Red Blood Cells.

Diosmin and bromelain are bioactive compounds of plant origin with proven beneficial effects on the human cardiovascular system. We found that diosmin and bromelain slightly reduced total carbonyls levels and had no effect on TBARS levels, as well as slightly increased the total non-enzymatic antioxidant capacity in the RBCs at concentrations of 30 and 60 µg/mL. Diosmin and bromelain induced a significant increase in total thiols and glutathione in the RBCs. Examining the rheological properties of RBCs, we found that both compounds slightly reduce the internal viscosity of the RBCs. Using the MSL (maleimide spin label), we revealed that higher concentrations of bromelain led to a significant decrease in the mobility of this spin label attached to cytosolic thiols in the RBCs, as well as attached to hemoglobin at a higher concentration of diosmin, and for both concentrations of bromelain. Both compounds tended to decrease the cell membrane fluidity in the subsurface area, but not in the deeper regions. An increase in the glutathione concentration and the total level of thiol compounds promotes the protection of the RBCs against oxidative stress, suggesting that both compounds have a stabilizing effect on the cell membrane and improve the rheological properties of the RBCs.

Indoxyl Sulfate Induces Oxidative Changes in Plasma and Hemolysate.

The deteriorating function of the kidneys in chronic kidney disease (CKD) is associated, among other things, with the retention of many unnecessary metabolic products in the body. Indoxyl sulfate (IS) belongs to the group of uremic toxins with a high protein binding affinity. Moreover, this compound can generate oxidative stress. We hypothesized that a high concentration of IS might induce oxidative changes in erythrocytes and plasma components, and could therefore contribute to CKD progression. In this study, we evaluated the influence of IS on the oxidative stress parameters in plasma and hemolysate. Moreover, as a result of the action of IS, we observed a decrease in the total antioxidant capacity and a change in the activity of catalase and superoxide dismutase in hemolysate and plasma. The obtained results indicate that IS induces oxidative damage to hemolysate and plasma components. Greater changes in the parameters of oxidative stress were observed in hemolysate than in plasma treated with indoxyl sulfate. The obtained results suggest that the increased concentration of IS in patients with chronic kidney disease may lead to a decrease in the lifespan of erythrocytes in their bloodstream.

Alterations in the Plasma and Red Blood Cell Properties in Patients with Varicose Vein: A Pilot Study.

The varicose vein results from the inefficient functioning of the valves in the lower limb veins, making the blood flow slow down and leading to blood stasis and hypoxia. This type of vein dysfunction might be a result of the development of oxidative stress. We compared oxidative stress markers in the plasma and erythrocytes obtained from peripheral veins and varicose veins in the same patients (glutathione, nonenzymatic antioxidant capacity (NEAC), catalase (CAT) and acetylcholinesterase (AChE) activity, thiols, thiobarbituric acid-reactive substance (TBARS), and protein carbonyls). We found a decrease in NEAC in the plasma obtained from the varicose veins compared to the peripheral veins. We detected a decrease in thiols in the plasma, hemolysate, and plasma membranes and increase in protein carbonyl compounds and TBARS levels in the varicose veins. These changes were accompanied by a decrease in CAT and AChE activity. For the first time, our results show changes in the plasma, erythrocyte membrane, and hemolysate protein properties in varicose vein blood in contrast to the plasma and erythrocytes in peripheral vein blood from the same patients. The increased oxidative stress accompanying varicose vein disease might result from the local inefficiency of the antioxidant defense system.

Uremic Toxins and Their Relation with Oxidative Stress Induced in Patients with CKD.

The presence of toxins is believed to be a major factor in the development of uremia in patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD). Uremic toxins have been divided into 3 groups: small substances dissolved in water, medium molecules: peptides and low molecular weight proteins, and protein-bound toxins. One of the earliest known toxins is urea, the concentration of which was considered negligible in CKD patients. However, subsequent studies have shown that it can lead to increased production of reactive oxygen species (ROS), and induce insulin resistance in vitro and in vivo, as well as cause carbamylation of proteins, peptides, and amino acids. Other uremic toxins and their participation in the damage caused by oxidative stress to biological material are also presented. Macromolecules and molecules modified as a result of carbamylation, oxidative stress, and their adducts with uremic toxins, may lead to cardiovascular diseases, and increased risk of mortality in patients with CKD.

Reactive Oxygen Species and Their Involvement in Red Blood Cell Damage in Chronic Kidney Disease.

Reactive oxygen species (ROS) released in cells are signaling molecules but can also modify signaling proteins. Red blood cells perform a major role in maintaining the balance of the redox in the blood. The main cytosolic protein of RBC is hemoglobin (Hb), which accounts for 95-97%. Most other proteins are involved in protecting the blood cell from oxidative stress. Hemoglobin is a major factor in initiating oxidative stress within the erythrocyte. RBCs can also be damaged by exogenous oxidants. Hb autoxidation leads to the generation of a superoxide radical, of which the catalyzed or spontaneous dismutation produces hydrogen peroxide. Both oxidants induce hemichrome formation, heme degradation, and release of free iron which is a catalyst for free radical reactions. To maintain the redox balance, appropriate antioxidants are present in the cytosol, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and peroxiredoxin 2 (PRDX2), as well as low molecular weight antioxidants: glutathione, ascorbic acid, lipoic acid, α-tocopherol, ß-carotene, and others. Redox imbalance leads to oxidative stress and may be associated with overproduction of ROS and/or insufficient capacity of the antioxidant system. Oxidative stress performs a key role in CKD as evidenced by the high level of markers associated with oxidative damage to proteins, lipids, and DNA in vivo. In addition to the overproduction of ROS, a reduced antioxidant capacity is observed, associated with a decrease in the activity of SOD, GPx, PRDX2, and low molecular weight antioxidants. In addition, hemodialysis is accompanied by oxidative stress in which low-biocompatibility dialysis membranes activate phagocytic cells, especially neutrophils and monocytes, leading to a respiratory burst. This review shows the production of ROS under normal conditions and CKD and its impact on disease progression. Oxidative damage to red blood cells (RBCs) in CKD and their contribution to cardiovascular disease are also discussed.

Structural component changes of erythrocytes caused by oxidative stress generated by indoxyl sulfate.

Indoxyl sulfate (IS) belongs to groups of uremic toxins binding to proteins. This compound may contribute to the generation of oxidative stress in chronic kidney disease (CKD) patients. We hypothesized that a high concentration of IS in the blood may induce structural changes of erythrocyte components and thus may contribute to CKD progression. In the present study, we evaluated the influence of IS on hemolysate and membrane proteins' conformational state, lipid membrane fluidity, and internal viscosity in erythrocytes. We examined thiols, carbonyl groups, peroxides, and TBARS levels in erythrocyte incubated with IS. The treatment of erythrocytes with IS led to increase in lipid membrane fluidity, decrease in the internal viscosity of the cells and the motion of the spin labels attached to hemolysate proteins. We did not observe conformational changes in plasma membrane proteins; however, in the plasma membranes of erythrocytes incubated with IS, a decrease in the content of thiol groups and increase in the carbonyls levels and peroxides and TBARS in comparison with the control was observed. The obtained results indicate that IS induces the oxidative damage of erythrocyte components. This may be an important factor that affects the functional properties of erythrocytes in CKD patients.

Alterations in the Properties of Red Blood Cells in Men with Coronary Artery Diseases after Comprehensive Cardiac Rehabilitation.

PURPOSE: Comprehensive cardiac rehabilitation (CCR) is a complex program aimed at improving the health status of patients with coronary artery disease (CAD), especially those who have been subjected to cardiac interventions (PCI and CABG).The aim of this study was to measure the changes in the properties of red blood cells (RBCs) in men with CAD after cardiac intervention and after participation in CCR program. METHODS: In this study, we have investigated the influence of the physical training-based CCR program in 12 men with CAD, after PCI or CABG. The characteristics of RBCs including the basic morphology of RBCs, the conformational state of RBC membrane protein and hemoglobin, acetylcholinesterase activity, membrane fluidity, the osmotic fragility, and thiol concentration in membrane and in hemolysate were measured. Ascorbate concentration and reduced glutathione were also determined. The analysis was performed in men, before and after participation in CCR. The properties of RBCs were observed in connection with the exercise test, and parameters were evaluated before, immediately after, and 1 hour after the exercise test. RESULTS: After CCR, a decrease in the mobility of erythrocyte membrane proteins was observed, which was accompanied by a decrease in lipid fluidity. In addition, immediately after the exercise test and 1 hour later, we measured a decrease in thiol level in hemolysate, but not in the plasma membrane. Unexpectedly, an increase in reduced glutathione concentration one hour after the exercise test after completing comprehensive cardiac rehabilitation was observed. CONCLUSION: CCR in men with CAD after cardiac intervention is connected with decreased membrane fluidity and decreased membrane protein mobility, which indicates that reduction of oxidative changes in these components occurs.

Combined effect of silver nanoparticles and aluminium chloride, butylparaben or diethylphthalate on the malignancy of MDA-MB-231 breast cancer cells and tumor-specific immune responses of human macrophages and monocyte-derived dendritic cells.

The aim of this study was to assess whether silver nanoparticles (AgNP) or selected cosmetic ingredients may modify functions of various immunocompetent cell populations. To this end, the effect of two AgNP (size of 15 nm or 45 nm), alone and in combination with aluminium chloride, butyl paraben, di-n-butyl phthalate or diethyl phthalate was assessed on: (1) migration and invasion of MDA-MB-231 human breast cancer cells; (2) M1/M2 polarization of phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages (M0) and (3) activation/maturation of monocyte-derived dendritic cells (DCs). The results of this study showed that neither any of the test chemicals alone nor the mixtures significantly changed the migration or invasion ability of MDA-MB-231 cells following, both 72-h and 21-day exposure. Analysis of the expression of marker genes for both M1 (IL-1B, CXCL9, TNF) and M2 (DCSIGN, MRC1) polarization revealed that the chemicals/mixtures did not activate M1/M2 differentiation of the M0 macrophages. In addition, no significant changes were observed in the expression of CD86, HLA-DR and CD54 surface markers and phagocytic activity of DCs following 48-h exposure to AgNP alone or in combination with test compounds. Our study suggests that AgNP alone or in combination with tested cosmetic ingredients do not alter function of immunocompetent cells studied.

Indoxyl Sulfate Generates Free Radicals, Decreases Antioxidant Defense, and Leads to Damage to Mononuclear Blood Cells.

Indoxyl sulfate (IS) is a uremic toxin that has been associated with inflammation and oxidative stress as well as with the progression of chronic kidney disease (CKD). IS is a protein metabolite that is concentrated in the serum of CKD patients. IS is a well-known uremic toxin, but there are very few reports on the effect of IS on cells including mononuclear cells (MNCs). We hypothesized that a high concentration of IS in CKD patients may induce changes in redox balance in the in vitro cells exposed. In the present study, we investigated the effect of IS on free radical production, antioxidant capacity, and protein damage in the mononuclear blood cells. As already determined, the concentrations (0.2 or 1 mM) of IS used in this study do not affect the survival rate of MNCs. For both the concentrations of IS, there was an increase in superoxide and nitric oxide and a release of other reactive oxygen species (ROS) inside the cells, as measured using fluorescent probes. However, an increase in other ROS as indicated by H2DCF-DA was found only for 1 mM of IS. Moreover, a decrease in the non-enzymatic antioxidant capacity and an increase in the superoxide dismutase activity after incubation of the cells with IS were observed. Furthermore, we found an increase in the levels of carbonyl compounds and peroxides in the cells treated with both the concentrations of IS. The obtained results show that IS induces oxidative stress and a decrease in antioxidant defense in cells leading to lipid and protein damage.

Alterations in conformational state of albumin in plasma in chronic hemodialyzed patients.

OBJECTIVE: In chronic hemodialyzed (CH) patients the balance between production of reactive oxygen species and antioxidant defense system is disturbed and shifted towards oxidative conditions. The properties of albumin in CH patients were studied before hemodialysis (HD) and post-HD. METHODS: Two oxidants were applied, organic t-butyl hydroperoxide (t-BOOH) and inorganic hydroperoxide (H2O2), for oxidation of albumin molecules. By comparison, albumin from healthy donors was also modified by both oxidants. The thiol content in albumin was determined by the Ellman method. Albumin properties were evaluated with the spin labelling technique using two covalently bound spin labels, maleimide (MSL) and iodoacetamide (ISL), and fatty acid spin probe, 16-doxylstearic acid (16-DS). RESULTS: A decrease in thiols level in HD albumin was greater than in control albumin. The t-BOOH modified the microenvironment at the binding site of MSL and ISL in control albumin molecules to a greater extent than hydrogen peroxide. Control albumin treated with t-BOOH and H2O2 showed an increase in the mobility of 16-DS. However, no changes were observed in albumin from CH patients treated with either of the oxidizing agents. CONCLUSION: Both oxidants induced strong conformational changes in albumin from healthy volunteers, but were less effective or ineffective in modification of albumin derived from CH patients. These results show that albumin from CH patients is highly modified in vivo and is not vulnerable to oxidation in the same way as normal albumin.

Inhibitory effect of silver nanoparticles on proliferation of estrogen-dependent MCF-7/BUS human breast cancer cells induced by butyl paraben or di-n-butyl phthalate.

In this study the effect of silver nanoparticles (AgNPs) on proliferation of estrogen receptor (ER)-positive human breast cancer MCF-7/BUS cells was assessed by means of in vitro assay. The cells were exposed in the absence of estrogens to AgNPs alone or in combination with aluminum chloride (AlCl3), butyl paraben (BPB) and di-n-butyl phthalate (DBPh). The results revealed that AgNPs at the non-cytotoxic concentrations (up to 2µg/mL) and AlCl3 (up to 500µM) did not induce proliferation of MCF-7/BUS cells whereas BPB and DBPh showed strong estrogenic activity with the highest effect at 16µM and 35µM, respectively. AgNPs inhibited the proliferation of the cells induced by DBPh, BPB or even with 17ß-estradiol (E2) during 6-day incubation in the absence of estrogens. ICI 182,780 (10nM), a known estrogen receptor (ER) antagonist, induced strong inhibitory effect. AgNPs also decreased transcription of the estrogen-responsive pS2 and progesterone receptor (PGR) genes but modulated expression neither of ERα nor ERß in MCF-7/BUS cells exposed to BPB, DBPh or E2 for 6h. Our results indicate that AgNPs may inhibit growth of breast cancer cells stimulated by E2 or estrogenic chemicals, i.e. BPB and DBPh.

Carbamylation and oxidation of proteins lead to apoptotic death of lymphocytes.

The apoptotic/necrotic changes in isolated human peripheral blood mononuclear cells (MNCs) subjected to hydrogen peroxide (H2O2), cyanate (NaOCN) and their combination were examined. The mitochondrial potential (ΔΨm), the activities of caspases (-2, -3, -6, -8 and -9) and the level of carbonyls and amino groups in proteins were determined and DNA fragmentation. Apoptotic or necrotic cells were identified by fluorescence microscopy using double staining with Hoechst 33258/propidium iodide. Treatment of MNCs with NaOCN (1 mmol/L and 2 mmol/L), alone and in combination with H2O2 (100 µmol/L), led to a significant decrease in the content of amine groups and a significant increase in the carbonyl level of MNCs in comparison with the control. Measurements taken at three time points (30, 60 and 150 min) showed a significant decrease in ΔΨm in MNCs incubated with H2O2, cyanate and their combination. The highest decrease in ΔΨm was observed after 150 min, when a combination of NaOCN and H2O2 was applied. We observed significant increases in the activities of caspases-2 and -3 in cells exposed to H2O2 and the combination of NaOCN and H2O2. An increase in caspase-2 but not in caspase-3 activity was noted in cells incubated with cyanate. A significant increase in caspase-9 activity in MNCs was observed in all arrangements of tested compounds in comparison with the control. In H2O2-treated cells, a higher level of necrotic cells was noted in comparison to apoptotic cells, whereas carbamylation led mainly to apoptotic cell death. The combination of cyanate and H2O2 increased the population of necrotic cells.

Transcriptomic analysis of the PI3K/Akt signaling pathway reveals the dual role of the c-Jun oncogene in cytotoxicity and the development of resistance in HL-60 leukemia cells in response to arsenic trioxide.

BACKGROUND: Arsenic trioxide (ATO) is a well-recognized antileukemic drug used for the treatment of newly diagnosed and relapsed acute promyelocytic leukemia (APL). A major drawback of therapy with ATO is the development of APL cell resistance, the mechanisms of which are still not clear. OBJECTIVES: The aim of this study was to investigate the role of the PI3K/Akt signaling pathway in ATOtreated human acute myeloid leukemia (HL-60) cells and in ATO-resistant clones. MATERIAL AND METHODS: The cytotoxicity of ATO was assessed using Trypan blue staining or a WST-1 reduction assay. The Akt phosphorylation level was measured by immunofluorescent staining and flow cytometry. Gene expression analysis was performed using real-time polymerase chain reaction (PCR). RESULTS: The clones derived by culturing for 8-12 weeks in the presence of 1.75, 2.5, and 5 µM ATO were characterized by high viability but a slower growth rate compared to the parental HL-60 cells. The flow cytometry analysis showed that in the parental cells the levels of p-Akt were undetectable or very low, and that ATO had no effect on the level of p-Akt in either the ATO-treated parental cells or the clones. The gene expression analysis revealed that some of the genes involved in the Akt pathway may play a key role in the induction of resistance to ATO, e.g., genes encoding cyclin D1 (CCND1), fork head box O1 (FOXO1), Jun oncogene (JUN), protein kinase C isoform B1 (PRKCB1), because their expression profiles were predominantly changed in the clones and/or the ATO-treated parental HL-60 cells. CONCLUSIONS: The overall results indicate that CCND1, FOXO1, and JUN may contribute to the induction of resistance to ATO, and that the C-Jun N-terminal kinase (JNK) signaling pathway may have greater significance than the phosphoinositide 3-kinase (PI3K)/Akt pathway in mediating the cytotoxic effects of ATO and the development of resistance to ATO in the HL-60 cell line.