The antitumor activity of Meconopsis horridula Hook, a traditional Tibetan medical plant, in murine leukemia L1210 cells.

BACKGROUND: Meconopsis horridula Hook (M. horridula) has been used as a traditional Tibetan medicine to relieve heat and pain as well as mobilize static blood, and it is recognized as a good treatment for bruises. This study is the first trial to evaluate the tumor inhibitory activity of M. horridula extract and its underlying mechanism in the hope of providing evidence to support the anticancer function of M. horridula. METHODS AND RESULTS: M. horridula extract was cytotoxic to L1210 cells in a dose- and time-dependent manner. SEM (scanning electron microscope) observation revealed obvious morphological changes in L1210 cells after M. horridula treatment. Flow cytometry analysis demonstrated that the extract dose-dependently induced early apoptosis. Additional apoptosis parameters, such as alterations in nuclear morphology and DNA damage, were also observed. Furthermore, M. horridula treatment induced G2/M arrest. M. horridula treatment significantly increased reactive oxygen species (ROS) production, suggesting that ROS are a key factor in M. horridula-induced apoptosis. Volatile constituent detection found 15 abundant chemicals in M. horridula, which may contribute to its anticancer effect. CONCLUSION: In conclusion, M. horridula extract induced L1210 cell apoptosis and inhibited proliferation through G2/M phase arrest, and ROS were involved in the process.

Effect of 9-cis retinoic acid and all-trans retinoic acid in combination with verapamil on P-glycoprotein expression in L1210 cells.

The development of the most common multidrug resistance (MDR) phenotype is associated with a massive overexpression of P-glycoprotein (P-gp) in neoplastic cells. In the current study, we used three L1210 cell variants: S cells - parental drug-sensitive cells; R cells - drug-resistant cells with P-gp overexpression due to selection with vincristine; T cells - drug-resistant cells with P-gp overexpression due to stable transfection with the pHaMDRwt plasmid, which encodes human full-length P-gp. Several authors have described the induction of P-gp expression/activity in malignant cell lines after treatment with all-trans retinoic acid (AtRA; ligand of retinoic acid nuclear receptors, RARs). An isomer of AtRA also exists, 9-cis retinoic acid, which is a ligand of both RARs and nuclear retinoid X receptors (RXRs). In a previous work, we described that the combined treatment of R cells with verapamil and AtRA induces the downregulation of P-gp expression/activity. In the current study, we studied the expression of RARs and RXRs in S, R and T cells and the effects of treatment with AtRA, 9cRA and verapamil on P-gp expression, cellular localization and efflux activity in R and T cells. We found that the overexpression of P-gp in L1210 cells is associated with several changes in the specific transcription of both subgroups of nuclear receptors, RARs and RXRs. We also demonstrated that treatment with AtRA, 9cRA and verapamil induces alterations in P-gp expression in R and T cells. Particularly, combined treatment of R cells with verapamil and AtRA induced downregulation of P-gp content/activity. In contrast, similar treatment of T cells induced slight increase of P-gp content without any changes in efflux activity of this protein. These findings indicate that active crosstalk between the RAR and RXR regulatory pathways and P-gp-mediated MDR could take place.

Acquisition of MDR phenotype by leukemic cells is associated with increased caspase-3 activity and a collateral sensitivity to cold stress.

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells that renders them unsusceptible to anti-neoplasic agents is one of the main causes of chemotherapy failure in human malignancies. The increased expression of P-glycoprotein (MDR1, P-gp, ABCB1) in tumor cells contributes to drug resistance by extruding chemotherapeutic agents or by regulating programmed cell death. In a study of MDR cell survival under cold stress conditions, it was found that resistant leukemic cells with P-gp over-expression, but not their sensitive counterparts, are hypersensitive to cold-induced cell death when exposed to temperatures below 4 °C. The transfection of parental cells with a P-gp-expressing plasmid makes these cells sensitive to cold stress, demonstrating an association between P-gp expression and cell death at low temperatures. Furthermore, we observed increased basal expression and activity of effector caspase-3 at physiological temperature (37 °C) in MDR cells compared with their parental cell line. Treatment with a caspase-3 inhibitor partially rescues MDR leukemic cells from cold-induced apoptosis, which suggests that the cell death mechanism may require caspase-3 activity. Taken together, these findings demonstrate that P-gp expression plays a role in MDR cell survival, and is accompanied by a collateral sensitivity to death induced by cold stress. These findings may assist in the design of specific therapeutic strategies to complement current chemotherapy treatment against cancer.

14-3-3 gamma is stimulated by IL-3 and promotes cell proliferation.

IL-3 plays important roles in the growth and survival of hematopoietic progenitor cells, processes modeled in studies of the IL-3-dependent cell line Ba/F3. To gain insights into molecular mechanisms governing cell fate, we examined the patterns of proteins up-regulated following stimulation of Ba/F3 cells with IL-3. Through two-dimensional electrophoresis and proteomics-based approaches, we identified 11 proteins. Of these, expression of 14-3-3gamma was significantly increased by IL-3 stimulation at both the transcriptional and translational levels. 14-3-3gamma overexpression in Ba/F3 cells abrogated dependence on IL-3 and was associated with activation of PI3K and MAPK signaling cascades, suggesting that the functions of 14-3-3gamma in normal hematopoietic progenitors are to promote survival and growth through the activation of distinct signaling pathways. Additionally, the up-regulation of Bax and Bad was seen with the ablation of 14-3-3gamma, resulting in cell death. These results indicate that deregulated expression of 14-3-3gamma may contribute to malignant transformation, possibly providing a new target for therapeutic intervention in hematopoietic neoplasms.

[Heterogeneity of cell population of lymphoma NK/Ly and leukemia L-1210 according to carbohydrate structure of cell surface: immunocytochemical analysis of lectin binding].

The heterogeneity of tumor cell populations according to binding of lectins from lentil (LcL), wheat germs (WGA), peanut (PNA) and concanavalin A was investigated on a model of murine Nemeth-Kellner lymphoma (NK/Ly) and leukemia L-1210. Bound lectins were detected by indirect immunochemical method using home obtained polyclonal antilectin antibodies and immunogold silver staining (IGSS) technique. Significant differences in binding of Con A were revealed between NK/Ly (67% Con A+) and L-1210 (7.2% Con A+) cells, while the differences in binding of other lectins with both types of tumor cells were not significant. A relatively high percentage of PNA+ cells was registered that can indicate a high degree of desialization of membrane glycoproteins.

The binding of human lactoferrin to mouse peritoneal cells.

Human iron-saturated Lf (FeLf), which was labeled with 125I or 50Fe, was found to combine with the membrane of mouse peritoneal cells (MPC) which consisted of 70% macrophages. The following experimental data suggested the involvement of a specific receptor. (a) The binding of FeLf to MPC reached a saturation point. (b) The binding of radioactive FeLf was inhibited by preincubating the cells with cold FeLf but not with human Tf, human aggregated and nonaggregated IgG, or beef heart cytochrome c (c) Succinylation and carbamylation of FeLf resulted in a loss of its inhibiting activity on the binding of radioactive FeLf. Removal of neuraminic acid from FeLf increased its inhibitory activity. (d) The ability of apoLf to inhibit the binding of FeLf to MPC was significantly lower than that of FeLf. The existence of a Lf receptor capable of concentrating Lf released from neutrophils on the membrane of macrophages could explain the apparent blockade of the release of iron from the reticuloendothelial system, which accounts for the hyposideremia of inflammation. A receptor for FeLf was also found on mouse peritoneal lymphocytes. The affinity constant of FeLf for both lymphocytes and macrophages was 0.9 X 12(6) liter/mol. Howerver, macrophages bound three times more FeLf molecules (20 X 10(6)) per cell than did lymphocytes (7 X 10(6)).

Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells.

A metabolic pathway of activated macrophages (M phi) involving oxidation of the guanido nitrogens of L-arginine is required for inhibition of growth and respiration of some target cells. The goal of this study was to identify the M phi metabolite(s) that induce these injuries. The stable products of the L-arginine pathway, NO2- and NO3-, were incapable of causing cytostasis under coculture conditions. However, NO2- became cytostatic upon mild acidification, which favors its transformation into nitrogen oxides of greater reactivity. This suggested that NO. (and/or NO2), recently identified as an M phi metabolite of L-arginine, could be a mediator. Authentic NO. caused cytostasis and respiratory inhibition in L1210 cells in a dose-dependent manner. The mitochondrial lesions caused by NO. were confined to complex 1 and 2, a pattern of injury identical to that seen after coculture with activated M phi. Inclusion of NO. scavenger systems prevented cytostasis from developing in M phi-L1210 cocultures. Thus, M phi-generated NO. can account for L-arginine-dependent cytostasis and respiratory inhibition.

Different expressions and DNA methylation patterns of lysophosphatidic acid receptor genes in mouse tumor cells.

OBJECTIVE: Lysophosphatidic acid (LPA) receptors act as several biological effectors through LPA, which is a bioactive phospholipid. Recently, aberrant expressions of LPA receptor genes due to DNA methylation have been detected in several tumor cells. In this study, we measured expression levels and DNA methylation status of LPA receptor genes in mouse tumor cells, LL/2 lung carcinoma, B16F0 melanoma, FM3A mammary carcinoma and L1210 leukemia cells, compared with normal tissues. METHODS: Total RNAs were extracted and RT-PCR analysis was performed. For DNA methylation status, bisulfite sequencing analysis was carried out, comparing outcomes with other tumor cells and normal tissues. RESULTS: The expressions of LPA1 gene were shown in LL/2, but not in B16F0, FM3A and L1210 cells. While the LPA2 gene was expressed in all 4 tumor cells, the LPA3 gene was unexpressed in them. The LPA1 and LPA3 unexpressed cells were highly methylated, although normal tissues were all unmethylated. The DNA methylation status was correlated with gene expression levels in cancer cells. CONCLUSION: The present results demonstrate that DNA methylation patterns of LPA receptor genes are dependent on cancer cell types, suggesting that LPA receptors may be new molecular targets for therapeutic approaches and chemoprevention.

Why the xanthine derivatives are used to study of P-glycoprotein-mediated multidrug resistance in L1210/VCR line cells.

There is generally well known that various xanthines occur frequently in natural products, e.g. black coffee, black tea, green tea, natural dyes etc. Xanthine molecules are good tolerated and metabolised by organisms. Moreover, natural xanthines and/or sythesized xanthines may recall a lot positive affects (hemorheologic properties, anti-inflammatory properties, tracheal smooth muscle relaxant, positive chronotropic and central nervous system-stimulating, etc.) and may even induce a quantity of changes on the molecular level (inhibition of cyclic nucleotide phosphodiesterases, inhibition of the synthesis of tumor necrosis factor (TNF-alpha), cellular Ca(2+) homeostasis, etc.). In our previous paper we showed that some xanthine derivatives (pentoxifylline and its derivatives) depress P-glycoprotein (P-gp) mediated multidrug resistance of the mouse leukemic cells. Other authors, first of all Sadzuka and co-workers, confirm this usefulness of long side substituted xanthines as biochemical modulators. However, the mechanism of molecular action of xanthine derivatives has not been clarified. One of the possible ways to chemosensitize the cancer cells is direct competiting in defence mechanism - inhibition of efflux pump (P-gp). Interaction of xanthine derivatives with binding site of P-gp is a question which could be solved by experiment; although, molecular modelling may clear up this matter. But, each dynamic and static program for molecular simulation of P-gp action is dividing on input variable, considering mechanistic view of insight drug transport.

Effect of thapsigargin on P-glycoprotein-negative and P-glycoprotein-positive L1210 mouse leukaemia cells.

Expression of drug-transporting P-glycoprotein (P-gp, an integral protein of the plasma membrane) in neoplastic cells confers multidrug resistance and also involves alteration of cell sensitivity to inhibitors of the sarco/endoplasmic reticulum calcium pump thapsigargin (Th). Mouse leukaemia L1210 cell sublines that overexpress P-gp due to selection with vincristine (R) or stable transfection with a gene encoding human P-gp (T) were less sensitive to Th than the parental cell line (S). Th at a concentration of 0.1 µmol/l did not induce alterations in the amount of P-gp mRNA in R or T cells (S cells did not contain any measurable amount of this transcript as assessed by RT-PCR) or in the amount of calnexin (CNX) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in all three cell sublines. However, when using a concentration of 10 µmol/l, Th decreases the amounts of CNX, GAPDH (in S, R and T cells) and P-gp (in R and T cells) mRNAs. In contrast to R and T cells (which contain abundant P-gp), S cells did not contain any P-gp detectable by the c219 antibody on a Western blot. Th at a concentration of 0.1 µmol/l induced a reduction in the amount of P-gp present in R and T cells, particularly in isoforms with higher molecular weights (i.e., mature fully glycosylated isoforms). Similar results were observed when Th was used at a concentration of 10 µmol/l. R and T cells contained lower levels of CNX than S cells. While Th at a lower concentration did not alter the levels of CNX in S, R or T cells, a higher concentration of this substance induced a measurable decrease in the amount of CNX. S, R and T cells did not differ with respect to GAPDH content, but Th induced a reduction in the amount of this protein in all cell sublines. More pronounced results were observed when Th was applied at a concentration of 10 µmol/l comparing with a concentration of 0.1 mmol/l. These changes may be involved together with the Th efflux activity of P-gp in Th-resistance associated with the P-gp-mediated multidrug resistance of R and T cells.

Cell surface tubulin in leukemic cells: molecular structure, surface binding, turnover, cell cycle expression, and origin.

We report here new characteristics of cell surface tubulin from a human leukemia cell line. These cells (CEM cells) possess tubulin that is readily iodinated on the surface of living cells, turns over at a rate identical to that of other surface proteins, and is present throughout the cell cycle. When removed with trypsin, it rapidly returns to the surface. Peptide mapping of iodinated surface tubulin indicates that it possesses a similar, but not identical, primary structure to total CEM and rat brain tubulin. Living CEM cells are able to bind specifically a subfraction of CEM tubulin from metabolically labeled high speed supernatants of lysed CEM cells. Surface tubulin is more basic than the total tubulin pool. The binding, which is saturable, is inhibited by unlabeled CEM high speed supernatants but not by excess thrice-cycled rat or bovine brain tubulin. Surface tubulin is also shown to bind to living nontransformed normal rat kidney cells but not to normal, circulating, mononuclear white cells. Activated lymphocytes produce a tubulin that binds to CEM cells. Since CEM tubulin was detected in the media of 6-h cultures of CEM cells, we must conclude that at least some of the surface tubulin comes from the media. We further conclude that these leukemic cells produce an unusual tubulin that may bind specifically to any membrane. The presence of iodinatable surface tubulin, however, appears to require both the production of a unique tubulin and the presence of a "receptor-like" surface binding component.

Transport and phosphorylation as factors in the antitumor action of cytosine arabinoside.

Survival of mice bearing different transplantable leukemias and treated with cytosine arabinoside was compared with uptake and subsequent phosphorylation of the drug in vitro. Capacity for nucleotide formation was correlated with response and is apparently an important determinant of drug sensitivity. Drug uptake, although apparently mediated, was similar in all cell lines.

Inhibition of growth of leukemia cells by enzymic folate depletion.

A new bacterial enzyme, designated as carboxypeptidase G(1), inhibited growth of the L5178Y and L1210 murine leukemias, as well as the Walker carcinoma and the human lymphoblastoid line (RPMI 4265), propogated in vitro. This enzyme hydrolyzes the glutamate moiety from both oxidized and reduced folate forms, and thus it may prove to be of value in creating rapid folate depletion in vivo.

Selective modification of glutathione metabolism.

Glutathione, a tripeptide thiol found in virtually all cells, functions in metabolism, transport, and cellular protection. It participates in the reduction of disulfides and other molecules, and conjugates with compounds of exogenous and endogenous origin. It protects cells against the destructive effects of reactive oxygen intermediates and free radicals. Modifications of glutathione metabolism may be achieved by administration of selective enzyme inhibitors, and also by giving compounds that increase glutathione synthesis. Such effects are useful in chemotherapy and radiation therapy and in protecting cells against the toxic effects of drugs, other foreign compounds, and oxygen.

Experimental reconsideration of the utility of serum starvation as a method for synchronizing mammalian cells.

Accurate cell-size determinations support the prediction that serum starvation and related whole-culture methods cannot synchronize cells. Theoretical considerations predict that whole-culture methods of synchronization cannot synchronize cells. Upon serum starvation, the fraction of cells with a G1-phase amount of DNA increased, but the cell-size distribution is not narrowed. In true synchronization, the cell-size distribution should be narrower than the cell-size distribution of the original culture. In contrast, cells produced by a selective (i.e. non-whole-culture) method have a specific DNA content, a narrow size distribution, and divide synchronously. The general theory leading to the conclusion that whole-culture methods for synchronization do not work implies that one can generalize these serum-starvation results to other cell lines and other whole-culture methods, to conclude that these methods do not synchronize cells.

3-(5-Nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline induces ROS-mitochondrial mediated death signaling and activation of p38 MAPK in murine L1210 leukemia cells.

Quinazoline derivatives are multitarget agents with a broad spectrum of biological activity. 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline (NTCHMTQ) is a new synthetically prepared derivative, which in our previous studies showed antiproliferative and apoptosis inducing activities towards murine L1210 leukemia cells. The aim of this study was to provide the insight into the molecular mechanism regulating NTCHMTQ-induced apoptosis in L1210 cells. The activity of caspases 3, 8 and 9, generation of reactive oxygen species (ROS), mitochondrial membrane potential changes, release of cytochrome c, degradation of PARP and activation of c-Jun N-terminal kinase 1/2 (JNK1/2), p38 MAPK and extracellular-regulated kinase 1/2 (ERK1/2) were investigated. NTCHMTQ induced production of ROS, activation of caspases 3 and 9, cytochrome c release, PARP cleavage and activation of p38 MAPK, with no activation of JNK1/2 and ERK1/2. Our resuls clearly demonstrate that NTCHMTQ induces apoptosis of L1210 leukemia cells through ROS-mitochondrial mediated death signaling and activation of p38 MAPK.

Curcumin down-regulates the multidrug-resistance mdr1b gene by inhibiting the PI3K/Akt/NF kappa B pathway.

Curcumin, a constituent of turmeric, has anti-inflammatory, anti-carcinogenic, and chemopreventive effects in several animal tumor models. The expression of P-glycoprotein (P-gp), encoded by the mdr gene, is often associated with multidrug resistance (MDR) to unrelated chemotherapeutic drugs in cancer cells. Here, we demonstrate that curcumin down-regulates P-gp expression in multidrug-resistant L1210/Adr cells. Transfection with a series of 5'-deleted constructs of the mdr1b gene promoter indicated that a proximal region between -205 and +42 of the sequence was responsible for the suppression of promoter activity by curcumin. This response might be associated with the inhibition of the phosphatidyinositol 3-kinase (PI3K)/Akt/nuclear factor-kappa B (NF-kappa B) signaling pathway by curcumin. Moreover, curcumin reversed the MDR of the L1210/Adr cells. Thus, curcumin can contribute to the reversal of the MDR phenotype, probably due to the suppression of P-gp expression via the inhibition of the PI3K/Akt/NF-kappa B signaling pathway.

Cisplatin and dimethyl sulfoxide react to form an adducted compound with reduced cytotoxicity and neurotoxicity.

PURPOSE: We present work that demonstrates that cisplatin reacts rapidly with dimethyl sulfoxide (DMSO) in solution and identify the structure and reactivity of the resulting compound. METHODS: Electrospray ionization-mass spectrometry (ESI-MS) and NMR were used to identify the chemical structure of compounds formed when DMSO reacts with cisplatin. We studied the reactivity of the identified compound with DNA. In vitro toxicity studies in neurons and cancer cells and in vivo toxicity studies in rats were used to determine both the cancer chemotherapeutic and toxic effects of the identified compound. RESULTS: Cisplatin binds rapidly with DMSO to form a DMSO adduct. The resulting compound has reduced ability to bind to double-stranded DNA both in vitro and in cells. This compound has reduced toxicity for cancer cells and neurons in vitro. In vivo nephrotoxicity studies show that the adducted compound has different nephrotoxicity and elimination characteristics than cisplatin. CONCLUSIONS: From this work, we conclude that dissolving cisplatin in DMSO results in formation of an adducted compound with different therapeutic and biological characteristics. Furthermore, future studies which propose using DMSO in combination with cisplatin for chemotherapeutic treatment in patients must be reconsidered. Due to the rapidity and nature of the reaction, DMSO and cisplatin should not be combined for patient treatment.

Overexpression of P-glycoprotein in L1210/VCR cells is associated with changes in several endoplasmic reticulum proteins that may be partially responsible for the lack of thapsigargin sensitivity.

L1210/VCR cells, which express an abundant amount of P-glycoprotein (P-gp), were found to be resistant to thapsigargin--an inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA). In the current paper, we have studied the possible differences among L1210 and L1210/VCR cells in expression of endoplasmic reticulum proteins involved in the regulation of calcium homeostasis and calcium-dependent processes. Amounts of mRNA encoding both calcium release channels (ryanodine receptor channels--RyR and IP3-receptor channels--IP3R) were found to be at similar levels in sensitive and resistant cells. However, mRNAs encoding IP3R1 or 2 were decreased in resistant cells cultivated in the presence of VCR (1.08 micromol/l), while mRNA encoding RyR remained unchanged. The amount of mRNA for SERCA2 was decreased in resistant cells when compared with sensitive cells. This decrease was more pronounced when resistant cells were cultivated in the presence of vincristine (VCR). Calnexin was found to be less expressed at the protein level in resistant as in sensitive cells. The level of mRNA encoding calnexin was decreased only when resistant cells were cultivated in the presence of VCR. Calnexin was found to be associated with immature P-gp in resistant cells. Thus, differences exist between sensitive and resistant cells in the expression of endoplasmic reticulum proteins involved in the control of intracellular calcium homeostasis or calcium-dependent processes. These changes may be at least partially responsible for the lack of sensitivity of resistant cells to thapsigargin.

Diverse histone modifications on histone 3 lysine 9 and their relation to DNA methylation in specifying gene silencing.

BACKGROUND: Previous studies of individual genes have shown that in a self-enforcing way, dimethylation at histone 3 lysine 9 (dimethyl-H3K9) and DNA methylation cooperate to maintain a repressive mode of inactive genes. Less clear is whether this cooperation is generalized in mammalian genomes, such as mouse genome. Here we use epigenomic tools to simultaneously interrogate chromatin modifications and DNA methylation in a mouse leukemia cell line, L1210. RESULTS: Histone modifications on H3K9 and DNA methylation in L1210 were profiled by both global CpG island array and custom mouse promoter array analysis. We used chromatin immunoprecipitation microarray (ChIP-chip) to examine acetyl-H3K9 and dimethyl-H3K9. We found that the relative level of acetyl-H3K9 at different chromatin positions has a wider range of distribution than that of dimethyl-H3K9. We then used differential methylation hybridization (DMH) and the restriction landmark genome scanning (RLGS) to analyze the DNA methylation status of the same targets investigated by ChIP-chip. The results of epigenomic profiling, which have been independently confirmed for individual loci, show an inverse relationship between DNA methylation and histone acetylation in regulating gene silencing. In contrast to the previous notion, dimethyl-H3K9 seems to be less distinct in specifying silencing for the genes tested. CONCLUSION: This study demonstrates in L1210 leukemia cells a diverse relationship between histone modifications and DNA methylation in the maintenance of gene silencing. Acetyl-H3K9 shows an inverse relationship between DNA methylation and histone acetylation in regulating gene silencing as expected. However, dimethyl-H3K9 seems to be less distinct in relation to promoter methylation. Meanwhile, a combination of epigenomic tools is of help in understanding the heterogeneity of epigenetic regulation, which may further our vision accumulated from single-gene studies.