L1210 Leukemia Cells Research Articles

The synthesis and molecular recognization of the polyamine transporter of hydrazine-modified diamine conjugates

A series of four novel hydrazine-modified diamine conjugates ( 7a- b, 8a- b) were synthesized and evaluated for cytotoxicity against Melanoma B16, α-difluoromethylornithine (DFMO)-treated B16, spermidine (SPD)-treated B16, Mouse leukemia L1210 and Hela cell lines. Both the DFMO-B16 and SPD-B16 experiments indicated that conjugates 7a- b and 8a- b could recognize the polyamine transporter (PAT) and enter the cells in part or in whole via PAT, although they were not as efficient as the reference, 9-anthracenemethyl homospermidine ( 1).

Apoptotic and autophagic responses to Bcl-2 inhibition and photodamage

Among the cellular responses to photodamage initiated by photodynamic therapy (PDT) are autophagy and apoptosis. While autophagy is a reversible process that can be both a survival and a death pathway, apoptosis is irreversible, leading only to cell death. In this study, we followed the fate of mouse leukemia L1210 cells after photodamage to the endoplasmic reticulum (ER) using a porphycene photosensitizer, where Bcl-2 was among the PDT targets. In wild-type cells, we observed a rapid wave of autophagy, presumed to represent the recycling of some damaged organelles, followed by apoptosis. Using shRNA technology, we created a Bax knockdown line (L1210/Bax(-)). In the latter cell line, we found a marked decrease in apoptosis after photodamage or pharmacologic inactivation of Bcl-2 function, but this did not affect PDT efficacy. Loss of viability was associated with a highly-vacuolated morphology consistent with autophagic cell death. Previous studies indicated pro-survival attributes of autophagy after low-dose PDT, suggesting that autophagy may be responsible for the 'shoulder' on the dose-response curve. It appears that attempts at extensive recycling of damaged organelles are associated with cell death, and that this phenomenon is amplified when apoptosis is suppressed.

Nitrogen-Containing Verticillene Diterpenoids from the Taiwanese Soft Coral Cespitularia taeniata

Extensive column chromatography of the ethanolic extract of the soft coral Cespitularia taeniata collected in Taiwan has resulted in the isolation of eight new nitrogen-containing verticillene diterpenes, designated cespitulactams D-K ( 1- 8), together with three known diterpenes, cespitulactams A ( 9) and B ( 10) and cespitularin F. In addition, one new derivative, 6- O-acetylcespitulactam F ( 11), was prepared from compound 3. The structures of these compounds were elucidated on the basis of spectroscopic analyses, especially HRMS and 2D NMR experiments. The cytotoxicity against human oral epidermoid carcinoma (KB) and murine L1210 leukemia cell lines and antimicrobial activities of 1- 8 and 11 were tested and evaluated. A biogenetic pathway for these novel diterpene alkaloids was also proposed.

Safety and Efficacy of Marqibo (Vincristine Sulfate Liposomes Injection, OPTISOME™) in for the Treatment of Adults with Relapsed or Refractory Acute Lymphoblastic Leukemia (ALL).

Introduction: Vincristine sulfate (VCR) is a lipophilic, cell-cycle specific, antineoplastic agent that inhibits cell division by specifically binding to tubulin in mitotic spindles. The activity of VCR is dose and time-dependent, but central and peripheral neuropthy prohibits its use beyond doses of 1.4 mg/m2 (capped at 2 mg). Marqibo is a formulation of VCR encapsulated in a sphingomyelin/cholesterol liposome (OPTISOME) with a longer half-life than VCR. In murine models using L1210 or P388 lymphoid leukemia cell lines, Marqibo demonstrated greater anti-tumor activity compared with VCR. Marqibo was therefore an appropriate agent to study in relapsed or refractory ALL.Methods: Two clinical trials have been completed. First, a phase II trial of single agent Marqibo given at 2 mg/m2 (no dose capping) every 2 weeks enrolled 16 patients (pts) [Thomas et al, Cancer 106:120, 2006]. A multicenter dose escalation phase I trial using weekly Marqibo (1.5, 1.825, 2.0, 2.25, 2.4 mg/m2) in combination with pulse dexamethasone followed. There were no restrictions on the number of prior therapies. Subjects with grade 2 or greater central or peripheral neuropathy were ineligible.Results: In total, 52 pts with relapsed or refractory ALL were treated in the two studies combined. Median age was 34 years (range, 19–77), 31 males/21 females, with a median number of prior salvage regimens of 2 (range, 1–3). Nine pts had confirmed Philadelphia positive disease (8 in the pre-imatinib era, 1 resistant to tyrosine kinase inhibitor therapy). All pts had previously received conventional VCR therapy. There were 8 complete remissions and 3 partial remissions for an overall response rate of 21% [95% CI, 11, 35]. An additional 12 pts (23%) achieved hematological improvements (e.g., clearance of marrow blasts, platelet transfusion independence). Five responders were able to undergo allogeneic stem cell transplantation following therapy with Marqibo. The maximum tolerated dose in the phase I trial was 2.25 mg/m2 owing to grade 3 motor neuropathy, grade 4 seizure, and grade 4 hepatotoxicity observed in 1 patient each at the 2.4 mg/m2 dose level. Grade 1–2 peripheral neuropathy was manageable with dose modifications and anti-neuralgia agents such as gabapentin. Commonly observed toxicities included febrile neutropenia, myelosuppression, abdominal pain, nausea, constipation, diarrhea, fatigue, and infusion-related pyrexia.Conclusions: In reviewing the clinical experience to date, Marqibo with or without pulse dexamethasone has provided clinically meaningful activity in heavily pre-treated adults with ALL. A multicenter trial of single agent Marqibo in the setting of second salvage therapy for adults with relapsed ALL is underway. A phase III multicenter trial of Marqibo in combination with standard chemotherapy for de novo elderly ALL is in the planning phase.

A relatively stable antifungal peptide from buckwheat seeds with antiproliferative activity toward cancer cells

An antifungal peptide with a molecular mass of approximately 4 kDa was isolated from buckwheat seeds by using ion-exchange chromatography on SP-Sepharose and Q-Sepharose, and gel filtration on Superdex peptide. The peptide was adsorbed on SP-Sepharose in 10 mM NH(4)OAc buffer (pH 4.5) and on Q-Sepharose in 10 mM NH(4)HCO(3) buffer (pH 9.4), and appeared to be highly purified after these two steps. It inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola with an IC(50) of 35 and 40 microM, respectively. Its antifungal activity was stable between 0 and 70 degrees C, and between pH 1.0/2.0 and 13. It inhibited proliferation of Hep G2 (hepatoma) cells, L1210 (leukemia) cells, breast cancer (MCF-7) cells, and liver embryonic WRL 68 cells with an IC(50) of 33, 4, 25, and 37 microM, respectively. On the other hand, the peptide was unable to evoke a mitogenic response from splenocytes or induce nitric oxide production by macrophages. It inhibited HIV-1 reverse transcriptase with an IC(50) of 5.5 microM.

A novel ribonuclease with antiproliferative activity from fresh fruiting bodies of the edible mushroom Hypsizigus marmoreus

An 18-kDa ribonuclease (RNase) with a novel N-terminal sequence was purified from fresh fruiting bodies of the mushroom Hypsizigus marmoreus. The purification protocol comprised ion exchange chromatography on DEAE cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose and Q-Sepharose and gel filtration by fast protein liquid chromatography on Superdex 75. The starting buffer was 10 mM Tris–HCl buffer (pH 7.2), 10 mM Tris–HCl buffer (pH 7.2), 10 mM NH 4OAc buffer (pH 5), 10 mM NH 4HCO 3 buffer (pH 9.4) and 200 mM NH 4HCO 3 (pH 8.5), respectively. Absorbed proteins were desorbed using NaCl added to the starting buffer. A 42-fold purification of the enzyme was achieved. The RNase was unadsorbed on DEAE cellulose, Affi-gel blue gel and CM-cellulose but adsorbed on Q-Sepharose. It exhibited maximal RNase activity at pH 5 and 70 °C. Some RNase activity was detectable at 100 °C. It demonstrated the highest ribonucleolytic activity (196 U/mg) toward poly C, the next highest activity (126 U/mg) toward poly A, and much weaker activity toward poly U (48 U/mg) and poly G (41 U/mg). The RNase inhibited [ 3H-methyl]-thymidine uptake by leukemia L1210 cells with an IC 50 of 60 μM.

Alternol inhibits proliferation and induces apoptosis in mouse lymphocyte leukemia (L1210) cells.

Alternol is a novel compound purified from the fermenting products by microorganisms named as Alternaria alternata var. monosporus from the bark of Yew. In this study, we tested its effect on mouse lymphocyte leukemia L1210 cells. Alternol was found to inhibit the proliferation and induce apoptosis in L1210 cells. When the cells were treated with Alternol, chromatin condensation and phosphatidylserine externalization were observed with the down-regulation of the pro-survival gene Bcl-2 and the activation of caspase-3, caspase-9, but not caspase-8. Moreover, exposure of cells to Alternol resulted in a significant increase in reactive oxygen species (ROS) and mitochondrial transmembrane potential (DeltaPsim) depolarization. Taken together, these results demonstrate that Alternol is a potent agent in inducing L1210 cells to apoptosis, which involve caspase activation and ROS generation.

Growth inhibitory effect of ethyl acetate‐soluble fraction of Cynara cardunculus L. in leukemia cells involves cell cycle arrest, cytochrome c release and activation of caspases

An extract of artichoke Cynara cardunculus L. (CCE) has been shown to exhibit antioxidant and antigenotoxic properties. In this study, the ability of CCE to inhibit the growth of L1210 and HL-60 leukemia cells was studied. Treatment of leukemia cells with a variety of concentrations of CCE (500-2500 microg/microL) for 24 h resulted in dose-dependent inhibition of leukemia cell growth. The cell growth inhibition was accompanied by G(0)/G(1) cell cycle arrest and by a loss of cells in S phase. Futhermore, apoptosis detected as a sub-G(0) cell population and apoptotic DNA fragmentation was observed. More detailed analyses of apoptosis induced by CCE in HL-60 cells revealed that apoptosis progressed through the caspase-9/-3 pathway, as release of cytochrome c, caspase-9/-3 activations and specific proteolytic cleavage of poly(ADP-ribose) polymerase. Taken together, the results suggest that CCE exerts an antiproliferative activity on leukemia cells and induces apoptosis of these cells through a mitochondrial/caspase dependent pathway.

Synthesis, antitumor activity and structure–activity relationships of a series of Ru(II) complexes

A series of octahedral Ru(II) polypyridyl complexes, [Ru(phen) 2L] 2+ (L = R-PIP and PIP = 2-phenylimidazo[4,5- f][1,10]phenanthroline) were synthesized and characterized by elementary analysis, 1H NMR and ES-MS, as well as UV–visible spectra and emission spectra. The antitumor activities of these complexes and their corresponding ligands were investigated against mouse leukemia L1210 cells, human oral epidermoid carcinoma KB cells, human promyelocytic leukemia cells (HL-60) and Bel-7402 liver cancer cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. It was found that the complexes [Ru(phen) 2L] 2+ (L = R-PIP) exert rather potent activities against all of these cell lines, especially for the KB cells (IC 50 = 4.7 ± 1.3 μM). The binding affinities of these Ru(II) complexes to CT-DNA (calf thymus DNA), as well as the DNA-unwinding properties on supercoiled pBR322 DNA were also investigated. The results showed that these Ru(II) polypyridyl complexes not only had an excellent DNA-binding property but also possessed a highly effective DNA-photocleavage ability. The structure–activity relationships and antitumor mechanism were also carefully discussed.

Resveratrol induces apoptosis, influences IL-6 and exerts immunomodulatory effect on mouse lymphocytic leukemia both in vitro and in vivo

Resveratrol, a dietary polyphenol found in grapes, has been proposed to act as a chemopreventive or anti-tumor agent in numerous epidemiologic studies. In this study, we investigate the antitumor and immunomodulation effects of resveratrol on mouse lymphocytic leukemia cells L1210 both in vitro and in vivo. Our finding indicates that resveratrol inhibits proliferation, induces apoptosis, and influences cell cycle of L1210 cells in a dose- and time-dependent manner in vitro. Furthermore, resveratrol can exert a dose-related regulatory effect on both innate and specific immune function to L1210-bearing mice. A normalization of CD4/CD8 ratios is noted as well as an enhancement of lymphocyte proliferation, NK cell activity and anti-SRBC titers. Interleukin-6 cellular content and release are suppressed by resveratrol as well as mRNA expression. In conclusion, the data provide new findings with respect to resveratrol mechanism of action to mouse lymphocytic leukemia.

E, E, E-1-(4-Arylamino-4-oxo-2-butenoyl)-3,5-bis(arylidene)-4-piperidones: A topographical study of some novel potent cytotoxins

A series of E, E, E-3,5-bis(arylidene)-1-(4-arylamino-4-oxo-2-butenoyl)-4-piperidones 4 (phenylidene) and 5 (4-nitrophenylidene) were prepared in order to explore the structural features of the N-acyl group which affects the cytotoxic potency. Evaluation toward human Molt 4/C8 and CEM T-lymphocytes revealed that many of the IC 50 figures were submicromolar and lower than melphalan. Marked inhibitory potencies toward murine leukemia L1210 cells were also noted. When evaluated against a panel of human tumor cell lines, three representative compounds in series 4 displayed selective toxicity to leukemia and colon cancer cell lines and were significantly more potent than the reference drug melphalan. Molecular modeling of representative compounds in both series 4 and the analogs, in which the configuration of the olefinic double bond was changed from E to Z (series 3), revealed that the torsion angles of the arylidene aryl rings and locations of the terminal arylaminocarbonyl groups may have contributed to the greater cytotoxic properties displayed in 3. Compounds 4c (3,4-dichlorophenylamino), d (4-methylphenylamino) and 5c (3,4-dichlorophenylamino), d (4-methylphenylamino) inhibited the activity of human N-myristoyltransferase by approximately 50% at concentrations of 50–100 μM. The compounds in series 4 and 5 were well tolerated in a short-term toxicity study in mice.

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

BackgroundPrevious 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.ResultsHistone 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.ConclusionThis 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.

Determinants of the Apoptotic Response to Lysosomal Photodamage

Studies with mouse leukemia L1210 cells revealed that selective lysosomal photodamage caused by any of three photosensitizing agents was followed by a gradual loss of the mitochondrial membrane potential (ΔΨm), release of cytochrome c into the cytosol, increased DEVDase activity (a measure of levels of caspase-3) and a limited apoptotic response. Similar effects were observed in the murine hepatoma 1c1c7 cell line. Immunofluorescence techniques employing 1c1c7 cells demonstrated the immediate release of the lysosomal enzyme cathepsin B following lysosomal photodamage. These studies suggest that the cytotoxic effects of lysosomal photodamage are initiated by released lysosomal proteases that either directly and/or indirectly activate caspases as a consequence of the induction of mitochondrial damage.

Isolation and characterization of an antifungal peptide with antiproliferative activity from seeds of Phaseolus vulgaris cv. ‘Spotted Bean’

A 7.3-kDa antifungal peptide with an N-terminal sequence exhibiting remarkable homology to defensins from other leguminous plants was isolated from Phaseolus vulgaris cv. 'Spotted Bean'. The isolation procedure involved ion exchange chromatography on O-diethylaminoethyl (DEAE) cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The peptide was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and SP-Sepharose. It exerted an antifungal action on Fusarium oxysporum and Mycosphaerella arachidicola. It inhibited mycelial growth in F. oxysporum with an IC(50) value of 1.8 microM. It suppressed [methyl-(3)H]-thymidine incorporation by leukemia L1210 cells and MBL2 cells with an IC(50) value of 4.0 and 9.0 microM, respectively. There was no effect on HIV-1 reverse transcriptase activity when the peptide was tested up to 0.1 mM.

Effects of Melophlins on Colony Formation of Chinese Hamster V79 Cells and IL-8 Production in PMA-stimulated HL-60 Cells

We have recently isolated four new melophlins P (1), Q (2), R (3), and S (4) together with seven known melophlins A (5), D (6), E (7), G (8), H (9), I (10), and O (11) from two marine sponges of the genus Melophlus collected in Palau. In this study, the influence of these compounds on the colony formation of Chinese hamster V79 cells and the production of IL-8 in PMA-stimulated HL-60 cells were examined. These 11 compounds did not show any effect on IL-8 production. The EC50 values of compounds 2, 3, 4, 5, 7, 9, 10, and 11 against V79 cells were 44.0, 13.3, 16.7, 27.2, 19.8, 8.5, 23.1, and 9.6 microM, respectively. The linear-chain-type compounds (1, 6, and 8) were not active against V79 cells at 50 microM. Although the growth inhibitory activity of these melophlins was not remarkable, some structure-activity relationships of these compounds against V79 and murine leukemia L1210 cells were observed.

PM 701 a Highly Selective Anti Cancerous Agent Against L1210 Leukemic Cells: In Vivo Clinical and Histopathological Study

PM 701 a Highly Selective Anti Cancerous Agent Against L1210 Leukemic Cells: In Vivo Clinical and Histopathological Study

Effects of Melophlins on Colony Formation of Chinese Hamster V79 Cells and IL-8 Production in PMA-stimulated HL-60 Cells

We have recently isolated four new melophlins P (1), Q (2), R (3), and S (4) together with seven known melophlins A (5), D (6), E (7), G (8), H (9), I (10), and O (11) from two marine sponges of the genus Melophlus collected in Palau. In this study, the influence of these compounds on the colony formation of Chinese hamster V79 cells and the production of IL-8 in PMA-stimulated HL-60 cells were examined. These 11 compounds did not show any effect on IL-8 production. The EC50 values of compounds 2, 3, 4, 5, 7, 9, 10, and 11 against V79 cells were 44.0, 13.3, 16.7, 27.2, 19.8, 8.5, 23.1, and 9.6 μM, respectively. The linear-chain-type compounds (1, 6, and 8) were not active against V79 cells at 50 μM. Although the growth inhibitory activity of these melophlins was not remarkable, some structure-activity relationships of these compounds against V79 and murine leukemia L1210 cells were observed.

Protein kinase C-alpha antagonizes apoptosis induction by histone deacetylase inhibitors in multidrug resistant leukaemia cells

Previous studies have documented that while several drug-resistant cells enter apoptosis upon treatment with histone deacetylase inhibitors (iHDACs), their drug-sensitive counterparts do not. In the present study, we have investigated at the molecular level why parental drug-sensitive tumor cells do not respond to Trichostatin A and suberoylanilide hydroxamic acid, two iHDACs that promote apoptosis in drug-resistant leukaemia cells. Taking murine leukaemia L1210 cells as a model, we have determined that: (i) PKC-α expression is more elevated in parental L1210 than in drug-resistant L1210/R cells, (ii) activation of PKC neutralizes iHDACs-mediated apoptosis in L1210/R cells, (iii) depletion of PKC in parental L1210 cells results in a positive response to iHDACs-mediated apoptosis, and (iv) transfection of a mutant constitutively active PKC-α form in L1210/R cells makes the cells refractory to apoptosis induction by iHDACs. These results allow us to conclude that activation/high expression of PKC-α protects parental drug-sensitive L1210 cells from iHDACs-mediated apoptosis. Thus, determination of PKC-α levels/activity in leukaemia seems to be relevant when choosing efficient chemotherapy protocols based on the use of apoptosis-inducing anticancer drugs.

The role of autophagy in the death of L1210 leukemia cells initiated by the new antitumor agents, XK469 and SH80

The phenoxypropionic acid derivative 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (XK469) and an analogue termed 2-{4-[(7-bromo-2-quinalinyl)oxy]phenoxy}propionic acid (SH80) can eradicate malignant cell types resistant to many common antitumor agents. Colony formation assays indicated that a 24 h exposure of L1210 cells to XK469 or SH80 inhibited clonogenic growth with CI(90) values of 10 and 13 micromol/L, respectively. This effect was associated with G(2)-M arrest and the absence of any detectable markers of apoptosis (i.e., plasma membrane blebbing, procaspase 3 activation, loss of mitochondrial membrane potential, and formation of condensed chromatin). Drug-treated cells increased in size and eventually exhibited the characteristics of autophagy (i.e., appearance of autophagosomes and conversion of microtubule-associated protein light chain 3-I to 3-II). The absence of apoptosis was not related to an inhibition of the apoptotic program. Cultures treated with XK469 or SH80 readily underwent apoptosis upon exposure to the Bcl-2/Bcl-x(L) antagonist ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate. Continued incubation of drug-treated cells led to a reciprocal loss of large autophagic cells and the appearance of smaller cells that could not be stained with Höechst dye HO33342, had a chaotic morphology, were trypan blue-permeable, and lacked mitochondrial membrane potential. L1210 cells cotreated with the phosphatidylinositol-3-kinase inhibitor wortmannin, or having reduced Atg7 protein content, underwent G(2)-M arrest, but not autophagy, following XK469 treatment. Hence, the therapeutic actions of XK469/SH80 with L1210 cultures reflect both the initiation of a cell cycle arrest as well as the initiation of autophagy.

Synthesis and In Vitro Biological Activity Evaluation of the Derivatives of Combretastatin A-4

Combretastatin A-4 (CA-4) is a potent anti-tumor agent, which strongly inhibits tubulin polymerization. A common problem is the inactivation of derivatives with cis-configurated ethylene bridges during storage. β-Lactam analogs of CA-4 were synthesized in order to avoid isomerization to E-forms. Moreover, B-ring modifications (3,4,5-trimethoxyphenyl ring, furan ring) were studied with regard to biological activity. Their cytotoxic effects were evaluated in vitro against L1210 leukemia cell. The derivatives might provide some rational basis for further optimal structural modification. Keywords: CA-4, Synthesis, Derivatives, Anti-tumor