Differential cytotoxic effects ofAnnona squamosa seed extracts on human tumour cell lines: Role of reactive oxygen species and glutathione

Annonaceous acetogenins are a new class of compounds that have been reported to have potent pesticidal, parasiticidal, anti-microbial, cell growth inhibitory activities. In this study, organic and aqueous extracts from the defatted seeds ofAnnona squamosa (custard apple) were tested on different human tumour cell lines for antitumoural activity. While organic and aqueous extracts induced apoptosis in MCF-7 and K-562 cells, they failed to do so in COLO-205 cells. Treatment of MCF-7 and K-562 cells with organic and aqueous extracts resulted in nuclear condensation, DNA fragmentation, induction of reactive oxygen species (ROS) generation and reduced intracellular glutathione levels. In addition downregulation of Bcl-2 and PS externalization by Annexin-V staining suggested induction of apoptosis in MCF-7 and K-562 cells by both the extracts through oxidative stress. On the contrary, COLO-205 cells showed only PS externalization but no change in ROS and glutathione levels. These observations suggest that the induction of apoptosis byA. squamosa extracts can be selective for certain types of cancerous cells     

Glutathione depletion restores the susceptibility of cisplatin-resistant chronic myelogenous leukemia cell lines to Natural Killer cell-mediated cell death via necrosis rather than apoptosis

We investigated the effect of intracellular glutathione (GSH) levels on Natural Killer-mediated apoptosis in cisplatin-resistant K562 cells. K562/B6 and K562/C9 are cisplatin-resistant K562 cells less susceptible to lysis by natural killer cells. Cisplatin-resistant K562 cells did not present the apoptotic pattern of DNA fragmentation as it was observed for their maternal counterparts. K562/B6 and K562/C9 cell lines produce 1.6- and 1.9-times more GSH than K562 cells. Treatment of both cell lines with D,L-buthionine-(S,R)-sulfoximine (BSO, a gamma-glutamyl cysteine synthetase inhibitor) decreased GSH levels and augmented cell death induced by NK cells via a necrotic rather than an apoptotic process. Proliferating cell nuclear antigen (PCNA) expression was elevated in cisplatin-resistant K562 subclones, and the reduction of GSH levels after treatment with BSO decreased the expression of PCNA. These results suggest that the GSH level affects the NK cell-mediated cell death of cisplatin-resistant K562 cells by inducing necrosis rather than apoptosis.     

Essential requirement of reduced glutathione (GSH) for the anti-oxidant effect of the flavonoid quercetin

We have analyzed the anti- or pro-oxidant effects of the flavonoid quercetin (QU) by evaluating, in U937 cell line, hydrogen peroxide (H(2)O(2)), superoxide anion reduced glutathione (GSH) content, mitochondrial membrane potential, DNA content, phosphatidylserine exposure on the outer face of the plasma membrane and cell viability. Polychromatic flow cytometry was used to evaluate in the same cells several functional parameters. For short periods of treatment QU exerted an anti-oxidant effect (decrease in H(2)O(2) levels), whereas for long periods it showed a pro-oxidant activity (increase in ). In these conditions, GSH content was reduced, and this correlated with a lack of anti-oxidant activity of QU, which in turn could be correlated with proapoptotic activity of this molecule. Thus, QU can exert different effects (anti-/prooxidant) depending on exposure times and oxidative balance, and in particular on stores of GSH.     

PKC inhibition is involved in trichosanthin-induced apoptosis in human chronic myeloid leukemia cell line K562

Trichosanthin (TCS), a type I ribosome-inactivating protein, induces cell death in various cell types including several tumor cell lines. However, the mechanism remains largely uncharacterized. In this study, we investigated the possible mechanism underlying its cytotoxicity by using human chronic myeloid leukemia cell line K562. We found that TCS induced apoptosis in K562 cells in a time- and concentration-dependent manner and can be blocked by caspase-3 inhibitors. Interestingly, TCS treatment induced a transient elevation in intracellular calcium concentration and a slow increase in reactive oxygen species production, while calcium chelators and antioxidants had no obvious effect on TCS-induced apoptosis, suggesting that calcium changes and reactive oxygen species may not be involved in TCS-mediated apoptosis in K562 cells. Instead we found that TCS partly inhibited PKC activity. Indeed, the PKC activator, PMA, inhibited while the PKC inhibitor, calphostin c, enhanced TCS-induced apoptosis. These PKC modulators had similar effects on TCS-induced cleavage of caspase-3, and caspase-3 inhibitors prevented calphostin c-enhanced apoptosis induced by TCS. In summary, we conclude that TCS induces apoptosis in K562 cells partly via PKC inhibition and caspase-3 activation.     

Maslinic acid induced apoptosis in bladder cancer cells through activating p38 MAPK signaling pathway

The present study determines the role of reactive oxygen species (ROS) production and calcium signaling evoked by the tumor necrosis factor-alpha (TNFα) on apoptosis in the human leukemia HL-60 and K562 cell lines. The results show that treatment of both cell lines cells with 10 ng/mL TNFα resulted in a rise in the percentage of apoptotic cells after 6 h of treatment. It was also observed that the administration of 10 ng/mL TNFα increased intracellular ROS production, as well as a time-dependent increase in caspase-8, -3, and -9 activities. The present results also show that the pretreatment with well-known antioxidants such as trolox and N-acetyl cysteine partially reduced the caspase activation caused by the administration of TNFα. The findings suggest that TNFα-induced apoptosis is dependent on alterations in intracellular ROS generation in human leukemia HL-60 and K562 cells.     

Toxicity mechanisms of onion (Allium cepa) extracts and compounds in multidrug resistant erythroleukemic cell line

Onion (Allium cepa) is being studied as a potential anticancer agent, but little is known regarding its effect in multidrug resistance (MDR) cells. In this work, the cytotoxicity of crude onion extract (OE) and fractioned extract (aqueous, methanolic and ethyl acetate), as well as some onion compounds (quercetin and propyl disulfide) were evaluated in Lucena MDR human erythroleukemic and its K562 parental cell line. The capacity of OE to induce apoptosis and/or necrosis in these cells, the possible participation of oxidative stress and DNA damage were also assessed. Similar sensitivities were obtained for both tumoral cells, however only OE caused significant effects in the cells. In K562 cells, a significant increase of apoptosis was verified while the Lucena cells experienced a significant increase of necrosis. An antioxidant capacity was verified for OE discarding oxidative damage. However, OE provoked similar significant DNA damage in both cell lines. Thus, the OE capacity to overcome the MDR phenotype suggests anti-MDR action of OE.     

Quercetin and anti-CD95(Fas/Apo1) enhance apoptosis in HPB-ALL cell line

Several malignant cell lines are resistant to CD95-(Apo1/Fas)-mediated apoptosis, even when the CD95 receptor is highly expressed. Sensitivity to CD95-induced apoptosis can be restored using different molecules. In this study, we showed that quercetin, a naturally occurring flavonoid, in association with the agonistic anti-CD95 monoclonal antibody, increases DNA fragmentation and caspase-3 activity in HPB-ALL cells. These cells have been selected for their known resistance to CD95-induced apoptosis. At molecular level, quercetin lowers the level of intracellular reactive oxygen species, reduces mitochondrial transmembrane potential, thereby leaving the expression of CD95 receptor unchanged.     

P-glycoprotein mediates resistance to A3 adenosine receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-n-methyluronamide in human leukemia cells

We studied effects of 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA) on apoptosis induction in the K562/Dox cell line, which overexpressed P-glycoprotein (P-gp, ABCB1, MDR1). We found that the K562/Dox cell line was significantly more resistant to Cl-IB-MECA than the maternal cell line K562, which did not express P-gp. Although both cell lines expressed the A3 adenosine receptor (A3AR), cytotoxic effects of Cl-IB-MECA were not prevented by its selective antagonist MRS1523 (3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate). Analysis of cell extracts revealed that the intracellular level of Cl-IB-MECA was significantly lower in the K562/Dox cell line than in the maternal cell line K562. The downregulation of P-gp expression using shRNA targeting ABCB1 gene led to increased intracellular level of Cl-IB-MECA and restored cell sensitivity to this drug. Similarly, valspodar (PSC-833), a specific inhibitor of P-gp, restored sensitivity of the K562/Dox cell line to Cl-IB-MECA with concomitant increase of intracellular level of Cl-IB-MECA in the resistant cell line, while it affected cytotoxicity of Cl-IB-MECA in the sensitive cell line only marginally. An enzyme based assay provided evidence for interaction of P-gp with Cl-IB-MECA. We further observed that cytotoxic effects of Cl-IB-MECA could be augmented by activation of extrinsic cell death pathway by Apo-2L (TRAIL) but not FasL or TNF-α. Our results revealed that Cl-IB-MECA induced an increase in expression of TRAIL receptors in K562 cells, which could sensitize cells to apoptosis induction via an extrinsic cell death pathway. Importantly, these effects were inversely related to P-gp expression. In addition, MRS1523 did not affect Cl-IB-MECA induced expression of TRAIL receptors.     

Guanosine supplementation reduces the antiproliferative and apoptotic effects of the IMPDH inhibitor gnidilatimonoein in K562 cells

IMPDH (inosine 5'-monophosphate dehydrogenase) is the rate-limiting enzyme in the de novo biosynthetic pathway of guanine nucleotides, which is usually up-regulated in human leukaemia cell lines. Our previous studies have classified gnidilatimonoein, isolated from Daphne mucronata, as an IMPDH inhibitor and a strong antiproliferative agent among several types of leukaemia cells. In the present study, we investigated the effects of gnidilatimonoein on intracellular GTP pool size and its link to differentiation and apoptosis of K562 cells. It was found that gnidilatimonoein inhibited cell proliferation and induced G0/G1 cell cycle arrest in K562 cells after 24 h exposure to a single dose of gnidilatimonoein (1.5 μM), while no significant effects were observed on unstimulated and phytohaemagglutinin-stimulated peripheral blood lymphocyte cells at the gnidilatimonoein dose (1.5 μM) used. Based on the morphological changes, Wright-Giemsa staining, benzidine assay and the expression of cell surface markers [GPIIb (glycoprotein IIb) and glycophorin A], as analysed by flow cytometry, we found that K562 cells had differentiated towards megakaryocytic lineage. In addition, gnidilatimonoein induced apoptosis among K562 cells based on Acridine Orange/ethidium bromide and annexin V/propidium iodide double-staining observations. These changes, which were abrogated by the addition of guanosine, became evident when the intracellular GTP level decreased to approx. 20-35% of the untreated control level. Based on these findings, it can be concluded that gnidilatimonoein induces differentiation and apoptosis in K562 cells through perturbation of GTP metabolism, as one of its routes of action.     

Induction of apoptosis in murine leukemia by diarylheptanoids from Curcuma comosa Roxb.

Diarylheptanoids, isolated from the rhizome of Curcuma comosa Roxb., have several biological activities including anti-oxidant and anti-inflammation. The present study investigated the effect of five diarylheptanoids isolated from C. comosa rhizome on the proliferation of murine P388 leukemic cells. Compound-092, (3S)-1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol, bearing a catechol moiety, was the most potent diarylheptanoid (IC₅₀ of 4 μM) in inhibiting P388 leukemic cell viability by causing DNA breakage and inducing apoptosis. Apoptotic cell death was characterized by the presence of chromatin condensation, formation of apoptotic bodies, DNA fragmentation, and externalization of plasma membrane phosphatidylserine. This compound increased caspase-3 activity about fivefold above the untreated control, decreased the intracellular reduced glutathione level, and impaired mitochondrial transmembrane potential. In the presence of Cu(II) ion, the compound exhibited a pro-oxidant activity causing DNA strand breakage and enhancing the anti-proliferative activity. The results provide evidence for the pro-oxidant activity of the diarylheptanoid bearing a catechol moiety in the induction of apoptosis in murine P388 leukemia.     

Salvianolic acid B, an antioxidant from Salvia miltiorrhiza, prevents Abeta(25-35)-induced reduction in BPRP in PC12 cells

Several lines of evidence support that beta-amyloid (Abeta)-induced neurotoxicity is mediated through the generation of reactive oxygen species (ROS) and elevation of intracellular calcium. Salvianolic acid B (Sal B), the major and most active anti-oxidant from Salvia miltiorrhiza, protects diverse kinds of cells from damage caused by a variety of toxic stimuli. In the present study, we investigated the effects of Sal B against beta-amyloid peptide 25-35 (Abeta(25-35))-induced neurotoxicity, focused mainly on the neurotoxic effects of Abeta(25-35) and the neuroprotective effects of Sal B on the expression of brain-pancreas relative protein (BPRP), which is a new protein and mainly expressed in brain and pancreas. Following exposure of PC12 cells to 20 microM Abeta(25-35), a marked reduction in the expression of BPRP was observed, accompanied with decreased cell viability and increased cell apoptosis, as well as increased ROS production and calcium influx. Treatment of the PC12 cells with Sal B significantly reversed the expression of BPRP and cell viability while it decreased ROS production and intracellular calcium. These data indicate that Abeta(25-35) decreases the expression of BPRP via enhanced formation of intracellular ROS and increased intracellular calcium, and that Sal B, as an anti-oxidant, protects against Abeta(25-35)-induced reduction in expression of BPRP through its effects on suppressing the production of ROS, calcium flux, and apoptosis. However, the role(s) of BPRP in AD and the definite mechanisms by which Sal B protects against Abeta(25-35)-induced reduction in the expression of BPRP require further study.     

Involvement of glutathione in the cytotoxicity of 9-isothiocyanatoacridine

Isothiocyanates (ITCs) are phytochemicals with promising cancer-preventive potential. To elucidate the mechanism of cytotoxicity of ITCs, their accumulation by cells and the role of intracellular glutathione, fluorescent 9-isothiocyanatoacridine (AcITC) was synthesized. The kinetic parameters for the reactions of AcITC with thiols were estimated and the influence of AcITC on human chronic myeloid leukemia cell line (K562) in regard to intracellular glutathione was studied. Cytotoxicity was evaluated by MTT assay, IC(50)=29.2 +/- 2.5 microM (48 h incubation). This acridine derivative was able to induce apoptosis of cells (morphological changes of cells and DNA fragmentation were observed) at least within certain dose that only decreased the level of intracellular glutathione, excessive doses (completely depleted intracellular pool of glutathione) induced necrosis rather than apoptosis. Our results indicated that apoptosis of leukemia cells induced by ITC is possible only if intracellular glutathione is not entirely depleted.     

Spontaneous mitochondrial membrane potential change during apoptotic induction by quercetin in K562 and K562/adr cells

Natural products from plants such as flavonoids are potential drugs to overcome multidrug resistance (MDR) in cancer treatments. However, their modes of action are still unclear. In this study, the effects of quercetin on mitochondrial membrane potential (DeltaPsim) change as well as quercetin's ability to induce apoptosis and inhibit Pgp-mediated efflux of 99mTc-MIBI in K562/adr cells were investigated. Quercetin exhibits cytotoxicity against erythroleukemic cells: IC50 are 11.0 +/- 2.0 micromol/L and 5.0 +/- 0.4 micromol/L for K562 and K562/adr, respectively. Quercetin induces cell death via apoptosis in both K562 and K562/adr cells and does not inhibit Pgp-mediated efflux of 99mTc-MIBI. Quercetin (10 micromol/L, 3 h) and etoposide (100 micromol/L, 24 h) induce similar levels of apoptosis in K562 and K562/adr cells. Quercetin induces an increase followed by a decrease in |DeltaPsim| value depending on its concentration. A decrease in the |DeltaPsim| value is associated with an increase in the percentage of early apoptotic cells. It is clearly shown that quercetin results in a spontaneous DeltaPsim change during apoptotic induction. Therefore, quercetin is potentially an apoptotic-inducing agent, which reacts at the mitochondrial level.     

Acadesine Kills Chronic Myelogenous Leukemia (CML) Cells through PKC-Dependent Induction of Autophagic Cell Death

CML is an hematopoietic stem cell disease characterized by the t(9;22) (q34;q11) translocation encoding the oncoprotein p210BCR-ABL. The effect of acadesine (AICAR, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) a compound with known antileukemic effect on B cell chronic lymphoblastic leukemia (B-CLL) was investigated in different CML cell lines. Acadesine triggered loss of cell metabolism in K562, LAMA-84 and JURL-MK1 and was also effective in killing imatinib-resistant K562 cells and Ba/F3 cells carrying the T315I-BCR-ABL mutation. The anti-leukemic effect of acadesine did not involve apoptosis but required rather induction of autophagic cell death. AMPK knock-down by Sh-RNA failed to prevent the effect of acadesine, indicating an AMPK-independent mechanism. The effect of acadesine was abrogated by GF109203X and Ro-32-0432, both inhibitor of classical and new PKCs and accordingly, acadesine triggered relocation and activation of several PKC isoforms in K562 cells. In addition, this compound exhibited a potent anti-leukemic effect in clonogenic assays of CML cells in methyl cellulose and in a xenograft model of K562 cells in nude mice. In conclusion, our work identifies an original and unexpected mechanism by which acadesine triggers autophagic cell death through PKC activation. Therefore, in addition to its promising effects in B-CLL, acadesine might also be beneficial for Imatinib-resistant CML patients.     

Effect of resveratrol on high glucose-induced stress in human leukemia K562 cells

Hyperglycemia, a symptom of diabetes mellitus, induces hyperosmotic responses, including apoptosis, in vascular endothelial cells and leukocytes. Hyperosmotic shock elicits a stress response in mammalian cells, often leading to apoptotic cell death. In a previous report, we showed that hyperosmotic shock induced apoptosis in various mammalian cells. Importantly, apoptotic biochemical changes (i.e., caspase-3 activation and DNA fragmentation) were blocked by antioxidant pretreatment during hyperosmotic shock-induced cell death. In the present study, we report that resveratrol, a phytoalexin present in grapes with known antioxidant and anti-inflammatory properties, attenuates high glucose-induced apoptotic changes, including c-Jun N-terminal kinase (JNK) activation and caspase-3 activation in human leukemia K562 cells. Experiments with the cell permeable dye, 2',7'-dichlorofluorescein diacetate (DCF-DA), an indicator of reactive oxygen species (ROS) generation, revealed that high glucose treatment directly increased intracellular oxidative stress, which was attenuated by resveratrol. In addition, high glucose-treated K562 cells displayed a lower degree of attachment to collagen, the major component of vessel wall subendothelium. In contrast, cells pretreated with resveratrol followed by high glucose exhibited higher affinity for collagen. The results of this report collectively imply the involvement of oxidative stress in high glucose-induced apoptosis and alterations in attachment ability. Moreover, resveratrol blocks these events by virtue of its antioxidant property.     

The oxidizing agent menadione induces an increase in the intracellular molecular oxygen concentration in K562 and A431 cells: Direct measurement using the new paramagnetic EPR probe fusinite

The intracellular molecular oxygen concentration in control and menadione-treated K562 (an erythroleukemic cell line that grows in suspension) and A431 (an epidermal carcinoma that grows in monolayer) cells was measured directly by using the new electron paramagnetic resonance (EPR) probe fusinite. Because the oxidizing agent menadione is known to damage mitochondria and the cytoplasmic membrane in other cell systems, before conducting measurements of oxygen concentration in K562 and A431 cells, it was necessary to establish injury in these systems as well. Consequently, morphological and flow cytometric analyses were conducted after menadione treatment. The data presented here show that the two cell lines are heavily damaged by menadione. Once this menadione-induced injury was demonstrated, measurements of oxygen concentration were carried out in both K562 and A431 cells. Treatment with this quinone induces a sharp increase in intracytoplasmic molecular oxygen in both cell lines (from about 1% to about 10 and 15% in K562 and A431 cells, respectively). In addition, to gain a more complete understanding of the effects of menadione on cells, the extracellular molecular oxygen concentration and the oxygen consumption rate were also measured in control and menadione-treated K562 cells. These measurements demonstrate that menadione treatment results in an increase in the extracellular oxygen concentration (from about 5% in controls to 15% in treated cells) as well as a decrease in the oxygen consumption rate (from about 10 ng O/min/10⁶ cells in controls to 3 ng O/min/10⁶ cells after menadione exposure). The importance of the new EPR probe fusinite in monitoring directly cellular functions in which oxygen is involved and the effects of menadione on cellular oxygen balance are discussed.     

Intracellular oxidation of hydroethidine: Compartmentalization and cytotoxicity of oxidation products

Hydroethidine (HE) is a blue fluorescent dye that is intracellularly converted into red-emitting products on two-electron oxidation. One of these products, namely 2-hydroxyethidium, is formed as the result of HE superoxide anion-specific oxidation, and so HE is widely used for the detection of superoxide in cells and tissues. In our experiments we exploited three cell lines of different origin: K562 (human leukemia cells), A431 (human epidermoid carcinoma cells), and SCE2304 (human mesenchymal stem cells derived from endometrium). Using fluorescent microscopy and flow cytometry analysis, we showed that HE intracellular oxidation products accumulate mostly in the cell mitochondria. This accumulation provokes gradual depolarization of mitochondrial membrane, affects oxygen consumption rate in HE-treated cells, and causes cellular apoptosis in the case of high HE concentrations and/or long cell incubations with HE, as well as a high rate of HE oxidation in cells exposed to some stimuli.     

Proteasome inhibition by quercetin triggers macroautophagy and blocks mTOR activity

The bioflavonoid quercetin has long been known to exert anti-tumor effects, although the underlying mechanisms remain unknown. Investigation of the potential interference of this anti-oxidant with the efficacy of cell stress-inducing anti-cancer drugs revealed extensive intracellular vacuolation induced by quercetin in epithelial cancer cells that led to cell cycle arrest and ensuing apoptosis. Accumulation of biomarkers of autophagy, including fluorescent autophagy markers and acidotropic dyes characterized these vacuoles as phagolysosomes. Prior to the formation of autophagosomes, an immediate and pronounced inhibition of the autophagy-controlling mTOR activity in quercetin-treated cancer cells occurred, accompanied by a marked reduction in the phosphorylation of the mTOR substrates 4E-BP1 and p70S6 kinase. Assessment of cellular proteasome activity revealed an effective and immediate inhibition of the activity of the proteasome by quercetin in cancer cells. In addition to the formation of autophagosomes, accumulation of poly-ubiquitinated protein aggregates was observed. Thus, proteasome inhibition by quercetin can be regarded as a major cause of quercetin-induced cancer cell death. These results suggest potential new applications for quercetin in cancer science and identify quercetin as an easy-to-handle agent to study proteasome activity, mTOR signaling and autophagy in human cancer cells for cell biological purposes.     

Astragalus membranaceus lectin (AML) induces caspase-dependent apoptosis in human leukemia cells

Objectives: Recently, plant lectins have attracted great interest due to their various biological activities such as anti‐cancer, anti‐fungal and anti‐viral activities. We have reported earlier concerning anti‐proliferation of human cancer cell lines by a galactose‐binding lectin (AML), from a Chinese herb, Astragalus membranaceus. In the present study, detailed investigations into the mechanism of such anti‐proliferation properties have been carried out. Materials and methods: Mechanism of apoptosis initiation in K562 cells by AML was investigated by morphology, flow cytometry and western blot analysis. Results: AML induced apoptosis in a caspase‐dependent manner in the chronic myeloid leukemia cell line, K562. Furthermore, we observed that cytotoxicity and apoptosis of K562 cells induced by AML were completely abolished in presence of lactose or galactose. Conclusions: Our results suggest that AML could act as a potential anti‐cancer drug.