Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of leukemic cells to arsenic trioxide

Cell culture, tissue chemistry and flow cytometry were used to determine whether antisense bcl-2 oligodeoxynucleotides enhanced the sensitivity of leukemia cells to arsenic trioxide. A combination of arsenic trioxide with antisense bcl-2 oligodeoxynucleotides inhibited cell growth, induced apoptosis and induced bcl-2 protein expression in K562 and NB4 leukemic cells more significantly than either arsenic trioxide or the oligodeoxynucleotides on their own (P<0.01). Thus, bcl-2 antisense oligodeoxynucleotides increase the sensitivity of leukemic cells to arsenic trioxide. Combined use of the two agents could be a novel and attractive strategy in leukemia treatment.     

Heme oxygenase-1 silencing increases the sensitivity of human osteosarcoma MG63 cells to arsenic trioxide

Arsenic trioxide (ATO) has been successfully used to treat leukemia and some solid malignant tumors. Our previous study regarding the effects of ATO on mesenchymal-derived human osteosarcoma MG63 cells showed that heme oxygenase-1 (HO-1) was strongly induced upon treatment with ATO. The present study sought to investigate the effect of silencing HO-1 on the sensitivity of osteosarcoma cells to ATO to determine the potential for therapeutic applications. Small hairpin RNA (shRNA)-mediated interference was used to silence HO-1 in MG63 cells. Viability, apoptosis, and intracellular reactive oxygen species (ROS) of the cells were assessed to evaluate the sensitivity of the cells to ATO as well as the potential mechanisms responsible. shRNA-mediated interference prevented the induction of HO-1, increased cell death, and increased intracellular ROS levels in MG63 cells upon treatment with ATO. Silencing HO-1 increased the susceptibility of MG63 cells to the chemotherapeutic drug ATO by enhancing intracellular accumulation of ROS. Our results suggest that the inhibition of HO-1 could improve the outcome of osteosarcoma treated with ATO.     

Various tolerances to arsenic trioxide between human cortical neurons and leukemic cells

Arsenic trioxide (As₂O₃) is very effective for treatment of acute promyelocytic leukaemia (APL) but little can pass through the blood-brain-barrier (BBB), which limits its use in the prevention and treatment of central nervous system leukaemia (CNSL). Before creating a non-invasive method to help As₂O₃’s access, the safe and effective therapeutic concentration of As₂O₃ in the CNS ought to be known. The changes of apoptosis biomarkers, [Ca²⁺]_i and PKC activity of both leukaemia cells and human cortical neurons, were monitored before and after being treated with As₂O₃ in vitro with laser confocal microscopy and Western blot. NSE concentration, the neuron invasive biomarker, was monitored by enzyme immunoassay (NSE-EIA). This study revealed that cortical neuron was more tolerable to As₂O₃ compared to NB4. 1.0 μmol / L As₂O₃ showed little influence on cortical neuron but effectively promoted apoptosis and induced differentiation of NB4.     

Various tolerances to arsenic trioxide between human cortical neurons and leukemic cells

Arsenic trioxide (As2O3) is very effective for treatment of acute promyelocytic leukaemia (APL) but little can pass through the blood-brain-barrier (BBB),which limits its use in the prevention and treatment of central nervous system leukaemia (CNSL). Before creating a non-invasive method to help As2O3 ’s access,the safe and effective therapeutic concentration of As2O3 in the CNS ought to be known. The changes of apoptosis biomarkers,[Ca2 ]i and PKC activity of both leukaemia cells and human cortical neurons,were monitored before and after being treated with As2O3 in vitro with laser confocal microscopy and Western blot. NSE concentration,the neuron invasive biomarker,was monitored by enzyme immunoassay (NSE-EIA). This study revealed that cortical neuron was more tolerable to As2O3 compared to NB4. 1.0 μmol / L As2O3 showed little influence on cortical neuron but effectively promoted apoptosis and induced differentiation of NB4.     

The inherent cellular level of reactive oxygen species: One of the mechanisms determining apoptotic susceptibility of leukemic cells to arsenic trioxide

Though reactive oxygen species (ROS) has been noticed to be involved in arsenic trioxide (As₂O₃)-induced apoptosis of tumor cells, its role in apoptosis signaling remained to be elucidated. The objective of this work was to explore the association of the inherent cellular ROS level with the susceptibility of the tumor cells to apoptosis induction by As₂O₃. Low concentration of As₂O₃ was administered to cultured leukemic cell lines NB4, U937, HL60 and K562. The difference in apoptotic sensitivity was displayed among four cell types. ROS probes were incubated with the cells in the absence of As₂O₃, and ROS was thus quantified relatively by flow cytometry. We manifested, in four cell types, the inherently existed difference in whole ROS quantity, and a positive correlation between the inherent ROS level and their apoptotic sensitivity to As₂O₃. Furthermore, by interference using a ROS producer, we demonstrated that an elevation of ROS level would sensitize the cells to As₂O₃-induced apoptosis. The results of the present work suggested that the inherent ROS level might be determinative in tumor cells for their apoptotic susceptibility to As₂O₃.     

The role of DNA polymerase beta in determining sensitivity to ionizing radiation in human tumor cells

Lethal lesions after ionizing radiation are thought to be mainly unrepaired or misrepaired DNA double-strand breaks, ultimately leading to lethal chromosome aberrations. However, studies with radioprotectors and repair inhibitors indicate that single-strand breaks, damaged nucleotides or abasic sites can also influence cell survival. This paper reports on studies to further define the role of base damage and base excision repair on the radiosensitivity of human cells. We retrovirally transduced human tumor cells with a dominant negative form of DNA polymerase β, comprising the 14 kDa DNA-binding domain of DNA polymerase β but lacking polymerase function. Radiosensitization of two human carcinoma cell lines, A549 and SQD9, was observed, achieving dose enhancement factors of 1.5–1.7. Sensitization was dependent on expression level of the dominant negative and was seen in both single cell clones and in unselected virally transduced populations. Sensitization was not due to changes in cell cycle distribution. Little or no sensitization was seen in G₁-enriched populations, indicating cell cycle specificity for the observed sensitization. These results contrast with the lack of effect seen in DNA polymerase β knockout cells, suggesting that polDN also inhibits the long patch, DNA polymerase β-independent repair pathway. These data demonstrate an important role for BER in determining sensitivity to ionizing radiation and might help identify targets for radiosensitizing tumor cells.     

A critical role of glutathione in determining apoptosis sensitivity and resistance in leukemia cells

In chemosensitive leukemias and solid tumors, anticancer drugs have been shown to induce apoptosis. Deficiencies in the apoptotic pathways may lead to chemoresistance. Here we report that glutathione (GSH) plays a critical role in activation of apoptosis pathways by CD95 (APO-1/Fas) or anticancer drugs. Upon treatment with anticancer drugs or CD95 triggering, CD95-resistant or Bcl-x(L) overexpressing CEM cells were deficient in activation of apoptosis pathways. CD95-resistant and Bcl-x(L) overexpressing CEM cells exhibited higher intracellular GSH levels in comparison to parental cells. Downregulation of GSH by L-buthionine-(S,R)-sulfoxime (BSO), a specific inhibitor of glutathione synthesis, reversed deficiencies in activation of apoptosis pathways by anticancer drugs or CD95. Interestingly, downregulation of GSH by BSO increased CD95 DISC formation in type I cells. In hybrids of CD95-resistant cells with sensitive cells and hybrids of overexpressing Bcl-x(L) cells with sensitive cells, the phenotype of apoptosis resistance was dominant. Also, in these hybrids, downregulation of GSH reversed CD95- and chemoresistance. We conclude that dominant apoptosis resistance depends, at least in part, on intracellular GSH levels, which may affect apoptosis signaling at different compartments, for example, the death receptor or mitochondria.     

Bcl‐2 hijacks the arsenic trioxide resistance in SH‐SY5Y cells

Aresenic trioxide (ATO) is proven to be active against leukaemia cells by inducing apoptosis and differentiation. Even though ATO could effectively induce remissions of leukaemia cells, the drug resistance was observed occasionally. To further dissect the mechanism of ATO resistance, we selected the ATO‐resistant SH‐SY5Y cells and found that Bcl‐2 controlled the sensitivity of ATO in SH‐SY5Y cells. We report that necroptosis, autophagy, NF‐ƘB and MAPK signalling pathway are not involved in ATO‐induced apoptosis. Moreover, the ATO‐resistant cells showed distinct mitochondrial morphology compared with that of ATO‐sensitive cells. Intriguingly, nude mice‐bearing ATO‐sensitive cells derived xenograft tumours are more sensitive to ATO treatment compared with that of ATO‐resistant cells. These data demonstrate that cancer cells can acquire the ATO‐resistance ability by increasing the Bcl‐2 expression.     

Studies of apoptosis of malignant lymphoma cells induced by arsenic trioxide

The present study investigated the effect of As(2)O(3)on malignant lymphoma cells. Cell apoptosis was detected by cell staining and TdT-mediated dUTP Nick-end Labelling (TUNEL). Cellular DNA and protein expression content were determined by immunohistochemistry and flow cytometry. It was found that 0.5-2.0 microm /l As(2)O(3)could inhibit cell growth, including Raji cells and lymphoma cells from patients, and induce apoptosis, such as condensed chromatin and nuclear fragmentation with intact cell membrane, i.e. apoptotic body. It was also found that the cells of the sub-G(1)phase increased significantly and bcl-2 gene expression was greatly downregulated. However, this effect was not observed for Jurkat cells under the same conditions. We concluded that As(2)O(3)at a range of 0.5-2.0 microm /l can inhibit the growth and induce apoptosis in malignant lymphoma cells, which may have therapeutic potential.     

Factors determining detergent resistance of erythrocyte membranes

The degree of detergent insolubility of cell membranes is a useful parameter to test the strength of lipid–lipid interactions relative to lipid–detergent interactions. Thus, solubility studies could give insights about lipid–lipid interactions relevant in domain formation. In this work we perform a detailed study of the solubilization of four different erythrocyte membrane systems: intact human and bovine erythrocytes, and human and bovine erythrocytes depleted in cholesterol with methyl-β-cyclodextrin. Each system was incubated with different concentrations of the non-ionic detergent Triton X-100, and the insoluble fraction was characterized by determining cholesterol and phosphorus content. A distinct solubilization behavior was obtained for the four systems, which was quantified by a “detergent resistance parameter” obtained from the fit of the solubility curves. In order to correlate these findings with membrane structural parameters, we quantify the degree of acyl chain order/rigidity of the original membranes by EPR spectroscopy, finding that detergent resistance is higher when acyl chains are more rigid. Regarding compositional properties, we found a good correlation between detergent resistance parameters and the total amount of cholesterol plus sphingomyelin in the original membranes. Our results suggest that a high degree of acyl chain packing is the determinant membrane factor for resistance to the action of Triton X-100 in erythrocytes.     

Hierarchy of in vitro sensitivity and resistance of tumor cells to cytotoxic effector cells,cytokines, drugs and toxins

Summary Drug resistance of tumor cells has led to the development of other therapeutic modalities including biological response modifiers, lymphokine-activated killer cells (LAK), and cytokines alone and in combination. The premise of these alternative modalities is that drug resistance can be overcome by other cytotoxic agents or cytotoxic effector cells. However, the relationship between tumor cell sensitivity to these different agents and the cytotoxicity caused by drugs is not known or well understood. Thus, understanding the relationship between these different systems of tumor cell cytotoxicity is essential for optimal therapeutic intervention. To this end, we compared the tumor cell cytotoxicity mediated by recombinant tumor necrosis factor (rTNF), cytotoxic effector cells (natural killer cells, monocytes, LAK cells), chemotherapeutic drugs, and microbial toxins. Human tumor cell lines sensitive and resistant to rTNF or drugs were used to evaluate the effectiveness of the other cytotoxic modalities. Sensitivity was considered as tumor cell cytotoxicity above 15% while resistance refers to that below 10%. Cell lines tested consisted of several histological types such as brain, lung, colon and ovarian tumors. In our experiments, cell lines made resistant to rTNF by coculture were also relatively resistant to unactivated monocytes and their supernatants. These lines were sensitive to all other methods tested including activated monocytes, natural killer and LAK cells, drugs, and toxins. The tumor lines naturally resistant to rTNF were found to have various degrees of sensitivity and resistance to these other systems. Upon the analysis of our data, a pattern emerged that suggested a hierarchy of sensitivity and resistance of the tumor cells to the cytotoxic mechanisms explored. From a majority of cell lines resistant to rTNF to a minority of lines resistant to LAK, we found an interesting gradation of sensitivity and/or resistance to the other cytotoxic modalities employed. The hypothesis of an underlying common mechanism of action within these systems is discussed.Supported in part by grant CA43 121 from the Department of Health and Human services, NIH, and NRSA clinical and fundamental immunology training grant A107 126, NIH (J. S.), and in part by a grant from the Concern Foundation, Los Angeles and a gift from the Boiron Foundation     

Pharmacogenomic determination of genes associated with sensitivity or resistance of tumor cells to curcumin and curcumin derivatives

Curcuma longa L. has long been used as a medicinal plant in traditional Chinese medicine against abdominal disorders. Its active constituent curcumin has anti-inflammatory, chemopreventive and cytotoxic properties. In the present investigation, we have analyzed the cytotoxic activity of curcumin and four derivatives. Among these compounds, ethoxycurcumintrithiadiazolaminomethylcarbonate was the most cytotoxic one. The curcumin-type compounds were not cross-resistant to standard anticancer drugs and were not involved in ATP-binding cassette transporter-mediated multidrug resistance. A combined approach of messenger RNA-based microarray profiling, COMPARE analyses and signaling pathway analyses identified genes as determinants of sensitivity and resistance to curcumin and specific signaling routes involved in cellular response to curcumin. These genes may be useful as biomarkers to develop individualized treatment options in the future. From a nutritional point of view, it is a thriving perspective to further investigate whether C. longa may be used as a spice to improve cancer therapy.     

Effect of arsenic trioxide on metallothionein and its conversion to different arsenic metabolites in hen liver

The metabolism of arsenic, its affinity to metallothionein (MT), its influence on selenium levels, and its biotransformation to different metabolites in the liver tissue of laying hens exposed to arsenic trioxide (As2O3) was investigated. The experiment was performed with two groups of hens fed for 19 d with either a standard diet or with the same diet enriched in arsenic (30 microg/g). The major findings were as follows: 1. After 19 d exposure, about 65% of the total liver As was found in the water-soluble phase (100,000g centrifuged supernatant). In liver supernatant, As binding was found mostly in the range of very low-molecular-weight proteins (Mr < 10,000). Although after exposure the amount of MT-like proteins increased, the As bound to it was only in trace amounts. The protein was identified by convential procedures as Zn,Cu-thionein with traces of selenium and arsenic. 2. Arsenic exposure resulted in almost unchanged Se levels regarding its tissue concentrations and distribution between supernatant and pellet, where about 10% of total Se was found in the supernatant. On the contrary, As exposure did affect Cd levels. Tissue Cd concentration was slightly diminished, but the percentage of tissue Cd found in the water-soluble phase was increased from 20% to 40%. 3. In methanol extracts of tissue and supernatant of the As-exposed group, only two arsenic compounds were detected, As(III) and dimethylarsinic acid (DMA), the latter prevailing.     

三氧化二砷对人胃腺癌细胞株SGC-7901细胞核及线粒体作用的实验研究

目的:研究三氧化二砷(As203)对人胃腺癌细胞株SGC-7901的生物效应及其对线粒体和半胱氨酸蛋白酶家族-3(caspase-3)的作用.方法:通过MTT比色实验检测不同浓度As2O3对该细胞株的生长抑制作用;经Hoechst 33258染色后用荧光显微镜观察细胞核的形态变化;经过细胞线粒体膜电位检测区分凋亡细胞与正常细胞,并经流式细胞仪分析;caspase-3吸光度检测法测定As2O3组caspase-3的活化程度.结果:As2O3明显抑制SGC-7901人胃腺癌细胞的生长,抑制作用的强度呈时间依赖性(方差分析,P<0.01);Hoechst 33258染色后荧光显微镜观察细胞核固缩碎裂边集呈强蓝色荧光;线粒体膜电位检测法,流式细胞仪检测法,caspase-3吸光度检测法均检测到胃腺癌细胞的凋亡.结论:As2O3破坏线粒体跨膜电位和激活caspase-3活性可能是As2O3诱导人胃腺癌SGC-7901细胞凋亡的重要机制.     

Plasmid-encoded resistance to arsenic and antimony.

Resistance determinants to the toxic oxyanionic salts of arsenic and antimony are found on plasmids of both gram-negative and gram-positive organisms. In most cases these provide resistance to both the oxyanions of +III oxidation state, antimonite and arsenite, and the +V oxidation state, arsenate. In both gram-positive and -negative bacteria, resistance is correlated with efflux of the anions from cells. The determinant from the plasmid R773, isolated from a gram-negative organism, has been studied in detail. It encodes an oxyanion-translocating ATPase with three subunits, a catalytic subunit, the ArsA protein, a membrane subunit, the ArsB subunit, and a specificity factor, the ArsC protein. The first two form a membrane-bound complex with arsenite-stimulated ATPase activity. The determinants from gram-positive bacteria have only the arsB and arsC genes and encode an efflux system without the participation of an ArsA homologue.     

Lineage-related sensitivity to apoptosis in human tumor cells undergoing hyperthermia

In this study the role of hyperthermia as an apoptotic trigger was analyzed in four human tumor cell lines: HL60, U937, DOHH₂, and K562. These cell lines were chosen because of their well known and different expression of bcl-2 and bcr-abl genes, the expression of which is known to be an antiapoptotic condition. HL60 and U937 cells were strongly susceptible to heat exposure, while DOHH₂ cells were weakly sensitive and K562 cells were resistant, thus suggesting a possible gene involvement in this type of programmed cell death. The mechanisms underlying this apoptosis were investigated by flow cytometry, agarose gel electrophoresis, and light and electron microscopy. A subdiploid peak and DNA laddering, both of which are parameters specifically correlated to programmed cell death, were present in HL60 and U937 and, even if less evident, in DOHH₂ cells undergoing hyperthermic treatment, and were absent in K562 cells. In addition, DNA single-strand cleavage was revealed by in situ nick translation, observed by confocal microscopy. Morphological analysis confirmed these results and revealed the typical chromatin changes, followed by the appearance of micronuclei and apoptotic bodies. Accepted: 26 November 1999