Vitamin E and rutin synergistically inhibit expression of vascular endothelial growth factor through down-regulation of binding activity of activator protein-1 in human promyelocytic leukemia (HL-60) cells

Reactive oxygen species (ROS) are strong inducers of the angiogenic hormone vascular endothelial growth factor (VEGF). Although, rutin (R) in combination with vitamin E (VE) has been shown to synergistically inhibit oxidative damage, it is unclear whether the combination of R and VE (R + VE) inhibits VEGF secretion in tumor cells. Using a human promyelocytic leukemia (HL-60) cell line, we showed that R in combination with VE synergistically decreased the expressions of VEGF protein and mRNA. We also demonstrated that R + VE significantly decreased the binding capacity of nuclear factor-activator protein-1 (AP-1) to the VEGF gene promoter and decreased the expression of c-Jun protein. Furthermore, we demonstrated that R + VE synergistically reduced insulin receptor substrate-1 (IRS-1) protein expression in HL-60 cells. The decrease of ROS was only partially associated with the decrease of VEGF secreted (r² = 0.12, P = 0.083). Thus, the present results indicate that R in combination with VE attenuates VEGF expression in HL-60 cells and that this effect is mediated by a decreased binding activity of AP-1 through down-regulation of protein expression of insulin-like growth factor 1 receptor (IGF1-R)/IRS-1, while the antioxidant activity of R + VE appears to play a minor role.     

Small molecule tolfenamic acid and dietary spice curcumin treatment enhances antiproliferative effect in pancreatic cancer cells via suppressing Sp1, disrupting NF-kB translocation to nucleus and cell cycle phase distribution

Combination of dietary/herbal spice curcumin (Cur) and COX inhibitors has been tested for improving therapeutic efficacy in pancreatic cancer (PC). The objective of this study was to identify agent with low toxicity and COX-independent mechanism to induce PC cell growth inhibition when used along with Cur. Anticancer NSAID, tolfenamic acid (TA) and Cur combination were evaluated using PC cell lines. L3.6pl and MIA PaCa-2 cells were treated with Cur (5–25 μM) or TA (25–100 μM) or combination of Cur (7.5 μM) and TA (50 μM). Cell viability was measured at 24–72 h posttreatment using CellTiter-Glo kit. While both agents showed a steady/consistent effect, Cur + TA caused higher growth inhibition. Antiproliferative effect was compared with COX inhibitors, Ibuprofen and Celebrex. Cardiotoxicity was assessed using cordiomyocytes (H9C2). The expression of Sp proteins, survivin and apoptotic markers (western blot), caspase 3/7 (caspase-Glo kit), Annexin-V staining (flow cytometry), reactive oxygen species (ROS) and cell cycle phase distribution (flow cytometry) was measured. Cells were treated with TNF-α, and NF-kB translocation from cytoplasm to nucleus was evaluated (immunofluorescence). When compared to individual agents, combination of Cur + TA caused significant increase in apoptotic markers, ROS levels and inhibited NF-kB translocation to nucleus. TA caused cell cycle arrest in G₀/G₁, and the combination treatment showed mostly DNA synthesis phase arrest. These results suggest that combination of Cur + TA is less toxic and effectively enhance the therapeutic efficacy in PC cells via COX-independent mechanisms.     

Recombinant human Erythropoietin enhanced the cytotoxic effects of tamoxifen toward the spheroid MCF-7 breast cancer cells

Erythropoietin (EPO) is widely used to treat anemia in patients undergoing chemotherapy for cancers. The main objective of this study was to investigate the effect of rHuEPO on the response of spheroid breast cancer, MCF-7, cells to tamoxifen treatment. The MCF-7 spheroids were treated with 10 mg/mL tamoxifen in combination with either 0, 10, 100 or 200 IU/mL rHuEPO for 24, 48 or 72 h. The viability of the MCF-7 cells was determined using the annexin-V, cell cycle, caspases activation and acridine orange/propidium iodide staining. rHuEPO-tamoxifen combination significantly (p greater than 0.05) increased the number of spheroid MCF-7 cells entering early apoptotic phase after 12 h and late apoptotic phase after 24 h of treatment; primarily the result of the antiproliferative effect tamoxifen. Tamoxifen alone significantly (p < 0.05) increased the caspase-3 and −9 activities in the spheroid MCF-7 cells by 200 to 550% of the control. Combination rHuEPO and tamoxifen produced much lesser effect on the caspase-8 activity. The rHuEPO in the combination treatment had concentration-dependently caused decrease in the caspase activities. rHuEPO-tamoxifen combination markedly increased MCF-7 cells entering the SubG0/G1 phase of the cell cycle by more than 500% of the control, while decreasing those entering the G2 + M and S phases by 50%. After 72 h, the combination treatment produced greater (p < 0.05) change in the SubG0/G1 phase than tamoxifen treatment alone. Morphologically, spheroid MCF-7 cells subjected to combination rHuEPO-tamoxifen treatment showed nuclear condensation and margination, cytoplasmic blebbing, necrosis, and early and late apoptosis. Thus, the study showed that rHuEPO-tamoxifen combination induced apoptosis in the spheroid MCF-7 cells. The apoptotic effect of the rHuEPO-tamoxifen combination treatment on the MCF-7 cells was greater than that produced by tamoxifen alone. The rHuEPO-tamoxifen treatment enhanced the caspase-independent apoptotic effects of tamoxifen on the spheroid MCF-7 cells.     

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl+ cells by endothelial nitric oxide synthase-mediated production of nitric oxide

Introduction  Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H₂O₂, which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. Materials and methods  Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl⁺ cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl⁺ CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl⁺ cells. Conclusion  NAC enhances imatinib-induced apoptosis of Bcr-Abl⁺ cells by endothelial nitric oxide synthase-mediated production of nitric oxide.     

Dichloroacetate Enhances Adriamycin-Induced Hepatoma Cell Toxicity In Vitro and In Vivo by Increasing Reactive Oxygen Species Levels

A unique bioenergetic feature of cancer, aerobic glycolysis is considered an attractive therapeutic target for cancer therapy. Recently, dichloroacetate (DCA), a small-molecule metabolic modulator, was shown to reverse the glycolytic phenotype, induce reactive oxygen species (ROS) generation, and trigger apoptosis in various tumor cells. In this work, the capacity of DCA to enhance Adriamycin (ADM) efficacy in hepatoma cells by modulating glucose metabolism and redox status was evaluated. Two human hepatoma (HCC-LM₃ and SMMC-7721) and a normal liver (LO₂) cell lines were treated with DCA or ADM alone, or in combination. Exposure of hepatoma cells to DCA/ADM combination resulted in significantly decreased cell viability and increased percentage of apoptotic cells as well as intracellular ROS levels, in comparison with treatment with DCA or ADM alone. However, simultaneous treatment with the thiol antioxidant N-acetylcysteine (NAC, 10 mmol/L) reduced the elevated ROS levels and protected hepatoma cells from the cytotoxic effects of DCA/ADM combination. L-buthionine-[S,R]-sulfoximine, an inhibitor of glutathione synthesis, enhanced hepatoma cell sensitivity to DCA/ADM combination. Interestingly, treatment with DCA/ADM combination did not significantly increase cytotoxicity in normal hepatocytes in comparison with the drugs administered individually. Finally, DCA reduced tumor growth and enhanced ADM efficacy on HCC-LM₃ hepatoma in mice. Overall, our data suggest that DCA enhances ADM cytotoxicity in hepatoma cells by increasing intracellular ROS levels and provide a strong biochemical rationale for the use of DCA in combination with ADM for treatment of hepatoma.     

Potent proapoptotic actions of dihydroartemisinin in gemcitabine-resistant A549 cells

Our recent studies have demonstrated the key roles of reactive oxygen species (ROS)-mediated caspase-8- and Bax-dependent apoptotic pathways in dihydroartemisinin (DHA)-induced apoptosis of A549 cells. This report is designed to investigate the proapoptotic mechanisms of DHA in gemcitabine (Gem)-resistant A549 (A549GR) cells. A549GR cells exhibited lower basal antioxidant capacity, higher level of basal ROS and intracellular Fe^2 + than Gem-sensitive A549 (A549) cells. In contrast to the sluggish ROS generation induced by Gem, DHA induced a rapid ROS generation within 30 min. Moreover, Gem induced similar ROS generation in both cell lines, while DHA induced more ROS generation in A549GR cells than in A549 cells. More importantly, after treatment with DHA, A549GR cells showed more potent induction in Bax activation, loss of mitochondrial membrane potential (ΔΨm), caspase activation and apoptosis than A549 cells. Furthermore, NAC pretreatment potently prevented DHA-induced ROS generation and loss of ΔΨm as well as apoptosis, and silencing Bax by shRNA or inhibition of one of caspase-3, -8 and -9 also significantly prevented DHA-induced apoptosis in both cell lines, indicating the key roles of ROS and Bax as well as the caspases. Collectively, DHA presents more potent proapoptotic actions in A549GR cells preferentially over normal A549 cells via ROS-dependent apoptotic pathway, in which Bax and caspases are involved.     

Reduction in hypoxia-reoxygenation-induced myocardial mitochondrial damage with exogenous methane

Albeit previous experiments suggest potential anti-inflammatory effect of exogenous methane (CH₄) in various organs, the mechanism of its bioactivity is not entirely understood. We aimed to investigate the potential mitochondrial effects and the underlying mechanisms of CH₄ in rat cardiomyocytes and mitochondria under simulated ischaemia/reperfusion (sI/R) conditions. Three-day-old cultured cardiomyocytes were treated with 2.2% CH₄-artificial air mixture during 2-hour-long reoxygenation following 4-hour-long anoxia (sI/R and sI/R + CH₄, n = 6-6), with normoxic groups serving as controls (SH and SH + CH₄; n = 6-6). Mitochondrial functions were investigated with high-resolution respirometry, and mitochondrial membrane injury was detected by cytochrome c release and apoptotic characteristics by using TUNEL staining. CH₄ admixture had no effect on complex II (CII)-linked respiration under normoxia but significantly decreased the complex I (CI)-linked oxygen consumption. Nevertheless, addition of CH₄ in the sI/R + CH4 group significantly reduced the respiratory activity of CII in contrast to CI and the CH₄ treatment diminished mitochondrial H₂O₂ production. Substrate-induced changes to membrane potential were partially preserved by CH₄, and additionally, cytochrome c release and apoptosis of cardiomyocytes were reduced in the CH₄-treated group. In conclusion, the addition of CH₄ decreases mitochondrial ROS generation via blockade of electron transport at CI and reduces anoxia-reoxygenation-induced mitochondrial dysfunction and cardiomyocyte injury in vitro.     

Effective and new potent drug combination: Histone deacetylase and Wnt/β-catenin pathway inhibitors in lung carcinoma cells

Lung cancer is the most commonly diagnosed cancer worldwide with a high mortality rate. In this study, the therapeutic effect of combination valproic acid and niclosamide was investigated on human lung cancer cell line. The effects of the compounds alone and combination therapy on cell viability were determined by sulforhodamine B and adenosine 5′-triphosphate viability assays. Flow cytometry was used to determine the cell death mechanism and DNA damage levels responsible for the cytotoxic effects of combination therapy. The presence of apoptosis in cells was supported by fluorescence microscopy and also by using inhibitors of the apoptotic signaling pathway. The increase in cellular reactive oxygen species (ROS) level in combination therapy was determined by H2DCFDA staining. The effect of N-acetyl-l -cysteine combination on ROS increase was investigated on cell viability. In addition, the expression levels of the proteins associated with epigenetic regulation and cell death were analyzed by Western blotting and gene expression levels were determined using real-time quantitative polymerase chain reaction.It was observed that the combination therapy showed a cytotoxic effect on the A549 lung cancer cells compared to the individual use of the inhibitors. The absence of this effect on normal lung cells revealed the presence of a selective toxic effect. When the mechanism of cytotoxicity is examined, it has been observed that combination therapy initiates the activation of tumor necrosis receptors and causes apoptosis by activated caspase. It was also observed that this extrinsic apoptotic pathway was activated on the mitochondrial pathway. In addition, ER stress and mitochondrial membrane potential loss associated with increased ROS levels induce cell death. When the data in this study were evaluated, combination therapy caused a dramatic decrease in cell viability by inducing the extrinsic apoptotic pathway in lung cancer cell line. Therefore, it was concluded that it can be used as an effective and new treatment option for lung cancer.     

Pyruvate secreted by human lymphoid cell lines protects cells from hydrogen peroxide mediated cell death

Reactive oxygen species (ROS) released from polymorphonuclear leukocytes and macrophages could cause DNA damage, but also induce cell death. Therefore inhibition of cell death must be an important issue for accumulation of genetic changes in lymphoid cells in inflammatory foci. Scavengers in the post culture medium of four lymphoid cell lines, lymphoblastoid cell lines (LCL), Raji, BJAB and Jurkat cells, were examined. Over 80% of cultured cells showed cell death 24 h after xanthine (X)/xanthine oxidase (XOD) treatment, which was suppressed by addition of post culture medium from four cell lines in a dose-dependent manner. H₂O₂ but not O^·-₂ produced by the X/XOD reaction was responsible for the cytotoxity, thus we used H₂O₂ as ROS stress thereafter. The H₂O₂-scavenging activity of post culture media from four cell lines increased rapidly at the first day and continued to increase in the following 2–3 days for LCL, Raji and BJAB cells. The scavenging substance was shown to be pyruvate, with various concentrations in the cultured medium among cell lines. Over 99% of total pyruvate was present in the extracellular media and less than 1% in cells. α-Cyano-4-hydroxycinnamate, a specific inhibitor of the H⁺-monocarbohydrate transporter, increased the H₂O₂-scavenging activity in the media from all four cell lines via inhibition of pyruvate re-uptake by cultured cells from the media. These findings suggest that lymphoid cells in inflammatory foci could survive even under ROS by producing pyruvate, so that accumulation of lymphoid cells with DNA damage is possible.     

Effects of microwave exposure and Gemcitabine treatment on apoptotic activity in Burkitt’s lymphoma (Raji) cells

Abstract

We investigated the effects of 1.8?MHz Global System for Mobile Communications (GSM)-modulated microwave (MW) radiation on apoptotic level and cell viability of Burkitt’s lymphoma (Raji) cells with or without Gemcitabine, which exhibits cell phase specificity, primarily killing cells undergoing DNA synthesis (S-phase). Raji cells were exposed to 1.8?GHz GSM-modulated MW radiation at a specific absorption rate (SAR) of 0.350?W/kg in a CO₂ incubator. The duration of the exposure was 24?h. The amount of apoptotic cells was analyzed using Annexin V-FITC and propidium iodide (PI) staining with flow cytometer. The apoptotic activity of MW exposed Raji cells was increased significantly. In addition, cell viability of exposed samples was significantly decreased. Combined exposure of MW and Gemcitabine increased the amount of apoptotic cells than MW radiation alone. Moreover, viability of MW?+?Gemcitabine exposed cells was lower than that of cells exposed only to MW. These results demonstrated that MW radiation exposure and Gemcitabine treatment have a synergistic effect on apoptotic activity of Raji cells.     

Trans Fatty Acids Enhanced Beta-Amyloid Induced Oxidative Stress in Nerve Growth Factor Differentiated PC12 Cells

The effects of trans fatty acids, elaidic acid (trans-9, C18:1) and linoelaidic acid (trans-9, trans-12 C18:2), at 20 or 40 μM in nerve growth factor differentiated PC12 cells with or without beta-amyloid peptide (Aβ) were examined. Elaidic acid treatment alone did not affect cell viability and oxidative injury associated markers (P > 0.05). However, co-treatments of elaidic acid and Aβ led to more reduction in mitochondrial membrane potential (MMP) and Na⁺-K⁺-ATPase activity, and more increase in DNA fragmentation and 8-hydroxydeoxyguanosine (8-OHdG) production than Aβ treatment alone (P < 0.05). Linoelaidic acid alone exhibited apoptotic and oxidative effects in cells via decreasing MMP and Na⁺-K⁺-ATPase activity, increasing reactive oxygen species (ROS) level, lowering glutathione content and glutathione peroxidase (GPX) activity (P < 0.05). The co-treatments of linoelaidic acid with Aβ further enhanced oxidative damage via enhancing the generation of ROS, nitrite oxide and 8-OHdG, elevating caspase-3, caspase-8 and nitric oxide synthase activities, as well as declining GPX, catalase and superoxide dismutase activities (P < 0.05). These results suggested that the interaction of linoelaidic acid and Aβ promoted oxidative stress and impaired mitochondrial functions in neuronal cells.     

The combination of berberine and irradiation enhances anti‐cancer effects via activation of p38 MAPK pathway and ROS generation in human hepatoma cells

Berberine, an isoquinoline plant alkaloid, has been known to generate a wide variety of biochemical and pharmacological effects. In order to elucidate the molecular mechanism for the berberine‐induced enhancement of radio‐sensitization, the human hepatoma HepG2 cells were treated with berberine combined with irradiation. The anti‐tumor effect of gamma radiation was found to be significantly enhanced by berberine. The evidences of apoptosis, such as apoptotic DNA fragmentation and annexin V staining, were observed in the cells treated with the combination of berberine and irradiation. Additionally, the levels of reactive oxygen species (ROS) and nitric oxide (NO) were apparently elevated in the combination system. The activations of p38, Bax, and caspase‐3 were also detected in the irradiated cells pretreated with berberine. The productions of ROS and annexin V staining in the cells treated with the combination of berberine and irradiation were significantly inhibited by the specific inhibitor of p38 MAPK, SB203580. The cell death induced by berberine alone or the combination of berberine and irradiation was suppressed by the anti‐oxidant, N‐acetyl cysteine (NAC). Taken together, the present results clearly indicate that the combination of berberine and gamma‐radiation enhance the anti‐cancer effects through the p38 MAPK pathway and ROS generation. J. Cell. Biochem. 107: 955–964, 2009. © 2009 Wiley‐Liss, Inc.     

Diabetes aggravates myocardial ischaemia reperfusion injury via activating Nox2‐related programmed cell death in an AMPK‐dependent manner

Cardiovascular diseases such as myocardial ischaemia have a high fatality rate in patients with diabetes. This study was designed to expose the crosstalk between oxidative stress and AMPK, a vital molecule that controls biological energy metabolism, in myocardial ischaemia reperfusion injury (I/RI) in diabetic rats. Diabetes was stimulated in rats using streptozotocin injection. Rats were separated on random into control, control + I/R, Diabetes, Diabetes + I/R, Diabetes + I/R + N‐acetylcysteine and Diabetes + I/R + Vas2870 groups. Myocardial infarct size was determined, and the predominant Nox family isoforms were analysed. In vitro, the H9C2 cells were administered excess glucose and exposed to hypoxia/reoxygenation to mimic diabetes and I/R. The AMPK siRNA or AICAR was used to inhibit or activate AMPK expression in H9C2 cells, respectively. Then, myocardial oxidative stress and programmed cell death were measured. Diabetes or high glucose levels were found to aggravate myocardial I/RI or hypoxia/reoxygenation in H9C2 cells, as demonstrated by an increase in myocardial infarct size or lactate dehydrogenase levels, oxidative stress generation and induction of programmed cell death. In diabetic rat hearts, cardiac Nox1, Nox2 and Nox4 were all heightened. The suppression of Nox2 expression using Vas2870 or Nox2‐siRNA treatment in vivo or in vitro, respectively, protected diabetic rats from myocardial I/RI. AMPK gene knockout increased Nox2 protein expression while AMPK agonist decreased Nox2 expression. Therefore, diabetes aggravates myocardial I/RI by generating of Nox2‐associated oxidative stress in an AMPK‐dependent manner, which led to the induction of programmed cell death such as apoptosis, pyroptosis and ferroptosis.     

Phytotoxicity of decomposing below-ground residues of Ageratum conyzoides: nature and dynamics of release of phytotoxins

We investigated the dynamics of phytotoxicity and the quantitative changes in phenolics in decomposing root residues of Ageratum conyzoides over a 60-day period. A set of four treatments, viz. residues alone (R), residues mixed into soils (R + S, R + 3S), and soil alone (S), were maintained and the quantitative changes in phytotoxicity were monitored on 1, 5, 10, 15, 20, 25, 30, 45, and 60 day after decomposition (DAD). The phytotoxicity during the decomposition process was evaluated in a laboratory bioassay against radish (Raphanus sativus). The phytotoxicity of R, R + S, and R + 3S treatments increased during initial period of decomposition (up to 20-DAD), and declined afterwards (i.e., at >20-DAD). In general, the phytotoxicity was in the order R > R + S > R + 3S treatments. It was paralleled by a similar trend of changes in the amounts of water-soluble phenolics that increased up to 20-DAD and thereafter declined. The amount of phenolics was in the order R > R + S > R + 3S. At 1-DAD, the amount of water soluble phenolics in R, R + S, and R + 3S treatments was 765.3, 594.5, and 251.3 μg/ml, respectively. It enhanced to 1,266.76, 845.5, and 416.0 μg/ml at 20-DAD. However, at 60-DAD, the amounts of phenolics in R, R + S, and R + 3S treatments was 149.93, 142.6, and 100.0 μg/ml, respectively. The study concludes that the phytotoxicity of below-ground residues of A. conyzoides changes during decomposition and was reduced upon the addition of soil to the residues.     

Perturbation of intracellular K<Superscript>+</Superscript> homeostasis with valinomycin promotes cell death by mitochondrial swelling and autophagic processes

Perturbation of cellular K⁺ homeostasis is a common motif in apoptosis but it is unknown whether a decrease in intracellular K⁺ alone is sufficient to replicate apoptotic hallmarks. We investigated, which mode of cell death is induced by decreasing the intracellular K⁺ concentration using valinomycin, a highly K⁺-selective ionophore. Valinomycin treatment induced mitochondrial swelling and minor nuclear changes in cell lines (BV-2, C6, HEK 293), and in primary mouse microglia and astrocytes. In the microglial cell line BV-2, we identified and quantified three phenotypes in valinomycin-exposed cells. The first and most prevalent phenotype (62 ± 2%) was characterized by swollen mitochondria and no chromatin condensation, and the second (25 ± 3%) by swollen mitochondria and slight chromatin condensation. Only the third phenotype (11 ± 4%) fulfilled criteria of apoptosis by having normal-sized mitochondria and strongly condensed chromatin. Valinomycin-induced swelling of mitochondria was not altered by the adenine nucleotide translocase inhibitor bongkrekic acid (BA), the pan caspase inhibitor Z-VAD-FMK, changing extracellular K⁺ or Cl⁻ concentrations, or the membrane-permeable Ca²⁺ chelator BAPTA-AM. Only co-exposure of cells to valinomycin and the Ca²⁺ ionophore ionomycin in high K⁺ Cl⁻-free extracellular solution suppressed mitochondrial swelling. Ionomycin alone caused shrinkage of mitochondria. Additionally, valinomycin promoted autophagic processes, which were further enhanced by preincubation with BA or with Z-VAD-FMK. Valinomycin-dependent chromatin condensation was inhibited by BA, Z-VAD-FMK, BAPTA-AM, and ionomycin. Our findings demonstrate that mitochondrial swelling and autophagy are common features of valinomycin-exposed cells. Accordingly, valinomycin promotes an autophagic cell death mode, but not apoptosis.     

Toxicity of xanthine oxidoreductase to malignant B lymphocytes

Reactive oxygen species (ROS) generated by xanthine oxidoreductase (XOR) were toxic to B lymphoma-derived Raji cells (positive for 8A monoclonal antibody, mAb). The sensitivity of these malignant cells to the hypoxanthine/XOR system was higher than that observed in peripheral human lymphocytes. The understanding of the mechanisms of cytotoxicity induced by XOR-produced ROS is essential in view of a possible clinical application. Cell death mostly had the feature of apoptosis and post-apoptotic necrosis and depended on the activity of XOR. Catalase, but not superoxide dismutase, protected cells from the toxicity of XOR, thus indicating that cell damage depended on the production of hydrogen peroxide. The toxicity of ROS was selectively targeted to malignant Raji cells by antibody-XOR conjugation, either directly, with an 8A-XOR conjugate, or indirectly, with an 8A mAb plus an anti-mouse IgG-XOR. Both direct and indirect immunotoxins induced apoptotic death to target cells in a dose-dependent manner. These conjugates showed no aspecific cytotoxicity in conditions very similar to the ex vivo treatment of cell suspension for bone marrow transplantation. Moreover, the prevalence of apoptotic death over necrosis may reduce the in vivo inflammatory response and its local and systemic consequences, thus becoming relevant in the construction of immunotoxins with therapeutic potential.     

Anticancer activities of genistein‐topotecan combination in prostate cancer cells

Prostate cancer is one of the leading causes of death in men aged 40 to 55. Genistein isoflavone (4′, 5′, 7‐trihydroxyisoflavone) is a dietary phytochemical with demonstrated anti‐tumour activities in a variety of cancers. Topotecan Hydrochloride (Hycamtin) is an FDA‐approved chemotherapy drug, primarily used for secondary treatment of ovarian, cervical and small cell lung cancers. This study was to demonstrate the potential anticancer efficacy of genistein‐topotecan combination in LNCaP prostate cancer cells and the mechanism of the combination treatment. The LNCaP cells were grown in complete RPMI medium, and cultured at 37°C, 5% CO₂ for 24–48 hrs to achieve 70–90% confluency. The cells were treated with varying concentrations of genistein, topotecan and genistein‐topotecan combination and incubated for 24 hrs. The treated cells were assayed for (i) post‐treatment sensitivity using MTT assay and DNA fragmentation, (ii) treatment‐induced apoptosis using caspase‐3 and ‐9 binding assays and (iii) treatment‐induced ROS generation levels. The overall data indicated that (i) both genistein and topotecan induce cellular death in LNCaP cells, (ii) genistein‐topotecan combination was significantly more efficacious in reducing LNCaP cell viability compared with either genistein or topotecan alone, (iii) in all cases, cell death was primarily through apoptosis, via the activation of caspase‐3 and ‐9, which are involved in the intrinsic pathway, (iv) ROS generation levels increased significantly with the genistein‐topotecan combination treatment. Treatments involving genistein‐topotecan combination may prove to be an attractive alternative phytotherapy or adjuvant therapy for prostate cancer.     

Attenuation of apoptotic DNA fragmentation by amiloride

Amiloride is a K⁺-sparing diuretic that effectively inhibits the Na⁺/H⁺ transporter in the plasma membrane of most mammalian cells. We have examined the effects of amiloride on the progression of apoptosis in HL-60 cells induced by camptothecin (CAM), cycloheximide (CHX), and 20 Gy gamma irradiation. Spectrofluorometric measurements on cell populations showed an inhibition of Na⁺/H⁺ transporter activity and a corresponding decrease in intracellular pH following treatment with amiloride alone, or in combination with the apoptosis-inducing agents. Flow cytometric cell cycle analysis, in combination with DNA strand break analysis, indicated that amiloride diminished endonuclease-mediated degradation of nuclear chromatin 3 h following treatment with CAM or CHX, and prevented degradation for 3 h following gamma radiation treatment. Apoptosis-associated DNA degradation was significantly greater for all three agents in the absence of amiloride. Protection from radiation-induced apoptosis was transient, since apoptotic subpopulations were observed, but still at a decreased level, 5 h following irradiation. Amiloride was as effective as zinc, an inhibitor of Ca²⁺/Mg²⁺-dependent endonucleases, in reducing or delaying the onset of endonuclease activity. Data presented show that effects of amiloride on membrane Na⁺/H⁺ transporter activity and intracellular pH can potentially affect apoptotic signaling cascades, leading to a retardation in the rate of progression to an apoptotic cell death. Results also point to the involvement of intracellular pH and Ca²⁺ in the regulation of apoptotic endonuclease activity, and the need for a functional Na⁺/H⁺ exchanger for the induction of apoptosis. J. Cell. Physiol. 175:59–67, 1998. © 1998 Wiley-Liss, Inc.     

Ferulic acid enhances the radiation sensitivity of lung and liver carcinoma cells by collapsing redox homeostasis: mechanistic involvement of Akt/p38 MAPK signalling pathway

Abstract

The major drawback of anticancer therapy is the development of resistance against drugs and radiation at the later phase of treatment which may lead to recurrences of the disease. Therefore, strategy was taken to enhance radiation sensitivity of lung (A549) and liver (HepG2) carcinoma cells by treatment with ferulic acid (FA) prior to irradiation. FA pre-treatment initially decreased reactive oxygen species (ROS) level in carcinoma cells which induced reductive stress and cytostasis. To overcome this stress, cellular mechanism increased the Keap1 level to down-regulate nuclear localisation of Nrf2 and its dependent antioxidant system. The antioxidant system reached the lowest level after 3 and 6?h of FA treatment in A549 and HepG2 cells respectively. As endogenous ROS were still being generated at same rate, ROS level was clearly higher than control which changed the reductive stress to oxidative stress. Exposure to γ-radiation in this condition further increased ROS level and caused radio-sensitisation of carcinoma cells. Combination of irradiation (IR) and FA activated mitochondrial apoptotic pathway and concomitantly inhibited the cell cycle progression and survival pathway over the IR group. Moreover, the combination treatment showed significant tumour regression, caspase 3 activation and nuclear fragmentation in tumour tissue compared to radiation alone. In contrast, FA pre-treatment protected peripheral blood mononuclear cells (PBMC) and normal lung fibroblast WI38 cells from radiation damage. Together, combination treatment offers effective strategy of killing cancer cells and demonstrates its potential for increasing the efficacy of radio-therapy.     

The changes of reactive oxygen species and glutathione by MG132, a proteasome inhibitor affect As4.1 juxtaglomerular cell growth and death

The proteasome inhibitor MG132 has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). Here, we evaluated the effects of MG132 on the growth and death of As4.1 juxtaglomerular cells in relation to ROS and glutathione (GSH) levels. MG132 inhibited the growth of As4.1 cells with an IC₅₀ of approximately 0.3–0.4 μM at 48 h and induced cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m), Bcl-2 decrease, activation of caspase-3 and -8, and PARP cleavage. MG132 increased intracellular ROS levels including O₂^? and GSH depleted cell numbers. N-acetyl cysteine (NAC, a well-known antioxidant) significantly decreased ROS level and GSH depleted cell numbers in MG132-treated As4.1 cells, along with the prevention of cell growth inhibition, cell death and MMP (ΔΨ_m) loss. NAC also decreased the caspase-3 activity of MG132. l-Buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) or diethyldithiocarbamate (DDC; an inhibitor of Cu/Zn-SOD) did not affect cell growth, death, ROS and GSH levels in MG132-treated As4.1 cells. Conclusively, MG132 reduced the growth of As4.1 cells via apoptosis. The changes of ROS and GSH by MG132 were involved in As4.1 cell growth and death.