Combination Chemotherapy of L1210 Tumors in Mice with Pretubulysin and Methotrexate Lipo-Oligomer Nanoparticles.

In the current study, nanoparticles containing the antimetabolite drug methotrexate (MTX) and the novel tubulin-binding drug pretubulysin (PT) were developed for combination chemotherapy. Polyelectrolyte complexes were formed based on ∼20 nm cationic nanomicelles of lipo-oligomer 454 with the anionic MTX at the molar ratio of 3:1, resulting in spherical nanoparticles with sizes of 150 nm (454 MTX). Particle formation in the presence of PT, which also interacts with 454, resulted in coloaded micelle complexes (454 PT+MTX) of 170 nm as demonstrated by transmission electron microscopy and dynamic light scattering measurements. Both drugs were incorporated to a high extent (∼85% for MTX, ∼70% for PT). Nanoparticles were stable in up to 20% serum and physiological NaCl solution. Cellular internalization of 454 PT+MTX into L1210 leukemia and KB cervix carcinoma cells was determined by confocal light scattering microscopy. The antitumor activity of the drug combination PT+MTX in both cell lines was strongly increased by drug formulation with 454 with IC50 values of PT+MTX decreasing 11-fold from 0.22 nM to 19 pM on L1210 cells and 6-fold from 2.8 to 0.48 nM on KB cervix carcinoma cells. Systemic treatment of NMRI nu/nu mice bearing subcutaneous L1210 tumors with 454 PT+MTX nanoparticles resulted in a more effective delay of tumor growth in comparison to the free drug combination of PT+MTX without 454. Importantly, nanoparticle formulation of PT+MTX with 454 increased the survival of mice by more than 100% compared to that of the buffer treated group and more than 40% compared to that of the free drug group.

Comparative Analysis of Bioactivity of the Russian-Made Antitumor Substances of the Nitrosourea Group.

We performed an in vivo comparative study of activity of three substances of the nitrosourea group produced in Russia. All substances demonstrated high antitumor activity against various solid and leukemic tumors. Aranosa significantly enhanced life duration in mice with leukemia (by 65-194%) and inhibited the growth of solid tumors (by 49-99.6%). Lisomustine and ormustine showed higher activity than aranose. Single administration of lisomustine increased life span of mice (by 22-114%) and resulted in cure of all animals in four models: lymphoblastic leukemia L-1210, lymphocytic leukemia P-388, Lewis lung carcinoma, and cervical cancer RShM-5. After ormustine treatment, full recovery was observed only in groups with lymphocytic leukemia P-388 and cervical cancer RShM-5. These findings attest to higher activity of lisomustine in the studied models.

Combined antitumoral effects of pretubulysin and methotrexate.

Pretubulysin (PT), a potent tubulin-binding antitumoral drug, and the well-established antimetabolite methotrexate (MTX) were tested separately or in combination (PT+MTX) for antitumoral activity in L1210 leukemia cells or KB cervix carcinoma cells in vitro and in vivo in NMRI-nu/nu tumor mouse models. In cultured L1210 cells, treatment with PT or MTX displays strong antitumoral effects in vitro, and the combination PT+MTX exceeds the effect of single drugs. PT also potently kills the MTX resistant KB cell line, without significant MTX combination effect. Cell cycle analysis reveals the expected arrest in G1/S by MTX and in G2/M by PT. In both cell lines, the PT+MTX combination induces a G2/M arrest which is stronger than the PT-triggered G2/M arrest. PT+MTX does not change rates of apoptotic L1210 or KB cells as compared to single drug applications. Confocal laser scanning microscopy images show the microtubule disruption and nuclear fragmentation induced by PT treatment of L1210 and KB cells. MTX changes the architecture of the F-actin skeleton. PT+MTX combines the toxic effects of both drugs. In the in vivo setting, the antitumoral activity of drugs differs from their in vitro cytotoxicity, but their combination effects are more pronounced. MTX on its own does not display significant antitumoral activity, whereas PT reduces tumor growth in both L1210 and KB in vivo models. Consistent with the cell cycle effects, MTX combined at moderate dose boosts the antitumoral effect of PT in both in vivo tumor models. Therefore, the PT+MTX combination may present a promising therapeutic approach for different types of cancer.

Involvement of mitochondrial dynamics in the antineoplastic activity of cisplatin in murine leukemia L1210 cells.

Leukemia is a type of hematopoietic stem cell malignant cloned disease with high mortality. Cisplatin-based chemotherapy is one of the most common treatments for leukemia. Similar to other chemotherapeutic agents, cisplatin resistance has become a serious issue in cancer therapy. In the present study, we investigated the role of mitochondrial dynamics in the antineoplastic activity of cisplatin in murine leukemia L1210 cells. Firstly, the L1210 cell line resistant to cisplatin (L1210/DDP) was established. Compared to its parental cell line, the IC50 value of cisplatin in the L1210/DDP cells was increased 10-fold. Mitofusins (Mfn1 and Mfn2), mitochondrial outer membrane fusion proteins, were markedly upregulated in the L1210/DDP cells, whereas the expression of fission protein Drp1 and inner membrane fusion protein OPA1 were not significantly altered. In addition, mitofusins were also upregulated in the parental L1210 cells subjected to cisplatin stress. To investigate the role of mitochondrial dynamics in the antineoplastic activity of cisplatin, the effect of mitochondrial division inhibitor (Mdivi)-1 on cisplatin­induced cell death, caspase-3 cleavage and ROS production was examined in L1210 cells. We found that 5 µM of Mdivi-1 efficiently attenuated cisplatin-induced cell death, caspase activation and intracellular ROS increase in L1210 cells. Our data indicated that mitochondrial dynamics play an important role in the antineoplastic activity of cisplatin, and mitofusin-mediated mitochondrial fusion may be involved in the process of cisplatin resistance in leukemia cells. Therefore, the present study revealed that mitochondrial dynamics may be a potential target used to improve the antineoplastic activity of cisplatin in leukemia in the future.

Preclinical Evaluation of the Short-Term Toxicity of 4-(N)-Docosahexaenoyl 2´, 2´- Difluorodeoxycytidine (DHA-dFdC).

PURPOSE: This study was designed to test the short-term toxicity of DHA-dFdC in a mouse model and its efficacy in a mouse model of leukemia at or below its repeat-dose maximum tolerated dose (RD-MTD). METHOD: A repeat-dose dose-ranging toxicity study was designed to determine the tolerability of DHA-dFdC when administered to DBA/2 mice by intravenous (i.v.) injection on a repeat-dose schedule (i.e. injections on days 0, 3, 7, 10, and 13). In order to determine the effect of a lethal dose of DHA-dFdC, mice were injected i.v. with three doses of DHA-dFdC at 100 mg/kg on days 0, 3, and 5 (i.e. a lethal-RD). The body weight of mice was recorded two or three times a week. At the end of the study, major organs (i.e. heart, liver, spleen, kidneys, lung, and pancreas) of mice that received the lethal-RD or RD-MTD were weighed, and blood samples were collected for analyses. Finally, DHA-dFdC was i.v. injected into DBA/2 mice with syngeneic L1210 mouse leukemia cells to evaluate its efficacy at or below RD-MTD. RESULTS: The RD-MTD of DHA-dFdC is 50 mg/kg. At 100 mg/kg, a lethal-RD, DHA-dFdC decreases the weights of mouse spleen and liver and significantly affected certain blood parameters (i.e. white blood cells, lymphocytes, eosinophils, and neutrophil segmented). At or below its RD-MTD, DHA-dFdC significantly prolonged the survival of L1210 leukemia-bearing mice. CONCLUSION: DHA-dFdC has dose-dependent toxicity, affecting mainly spleen at a lethal-RD. At or below its RD-MTD, DHA-dFdC is effective against leukemia in a mouse model.

Comparative studies of oxaliplatin-based platinum(iv) complexes in different in vitro and in vivo tumor models.

Using platinum(iv) prodrugs of clinically established platinum(ii) compounds is a strategy to overcome side effects and acquired resistances. We studied four oxaliplatin-derived platinum(iv) complexes with varying axial ligands in various in vitro and in vivo settings. The ability to interfere with DNA (pUC19) in the presence and absence of a reducing agent (ascorbic acid) was investigated in cell-free experiments. Cytotoxicity was compared under normoxic and hypoxic conditions in monolayer cultures and multicellular spheroids of colon carcinoma cell lines. Effects on the cell cycle were investigated by flow cytometry, and the capacity of inducing apoptosis was confirmed by flow cytometry and Western blotting. The anti-cancer activity of one complex was studied in vivo in immunodeficient and immunocompetent mice, and the platinum levels in various organs and the tumor after treatment were quantified. The results demonstrate that modification of the axial ligands can improve the cytotoxic potency. The complexes are able to interfere with plasmid DNA, which is enhanced by co-incubation with a reducing agent, and cause cell cycle perturbations. At higher concentrations, they induce apoptosis, but generate only low levels of reactive oxygen species. Two of the complexes increase the life span of leukemia (L1210) bearing mice, and one showed effects similar to oxaliplatin in a CT26 solid tumor model, despite the low platinum levels in the tumor. As in the case of oxaliplatin, activity in the latter model depends on an intact immune system. These findings show new perspectives for the development of platinum(iv) prodrugs of the anticancer agent oxaliplatin, combining bioreductive properties and immunogenic aspects.

Effects of the combined arginase and canavanine treatment on leukemic cells in vitro and in vivo.

It was previously demonstrated in in vitro experiments that canavanine (Cav), a natural toxic arginine analogue of plant origin, is a promising candidate for augmenting the antineoplastic effects of arginine starvation. We demonstrated herein that recombinant human arginase, an arginine degrading enzyme, abrogated growth and significantly increased Cav cytotoxicity toward cultured L1210 murine leukemic cells. Cav co-treatment further reduced cells viability in a time-dependent manner and significantly promoted apoptosis induction. In the pilot study we also evaluated for the first time the potential toxicity of the combined arginine deprivation and Cav treatment in healthy mice. Administration of Cav alone or in combination with pegylated cobalt-containing human arginase (Co-hARG) did not evoke any apparent toxic effects in these animals, with no significant behavioural and survival changes after several weeks of the treatment. The therapeutic effects of the combination of Co-hARG and Cav were provisionally evaluated on the highly aggressive murine L1210 leukemia, which is semi-sensitive to arginine deprivation as a monotreatment. Combination of two drugs did not result in significant prolongation of the survival of leukemia-bearing mice. Thus, we have shown that the proposed combinational treatment is rather non-toxic for the animals. It has to be further evaluated in animal studies with alternative tumor models and/or drug doses and treatment modalities.

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.

Preferential enlargement of leukemia cells using cytoskeletal-directed agents and cell cycle growth control parameters to induce sensitivity to low frequency ultrasound.

Sonodynamic therapy (SDT) is a form of ultrasound therapy that has been shown to preferentially damage malignant cells based on the relatively enlarged size and altered cytology of neoplastic cells in comparison to normal cells. This study sought to determine whether cytoskeletal-directed agents that either disrupt (cytochalasin B and vincristine) or rigidify (jasplakinolide and paclitaxel) microfilaments and microtubules, respectively, affect ultrasonic sensitivity. U937 human monocytic leukemia cell populations were treated with each cytoskeletal-directed agent alone, and then sonicated at 23.5 kHz under relatively low power and intensity (20-40 W; 10-20 W/cm(2)), or at 20 kHz using moderate power and intensity (60 W; 80 W/cm(2)). In addition, human leukemia lines U937, THP1, K562, and Molt-4, and the murine leukemia line L1210 were sonicated using pulsed 20 kHz ultrasound (80.6 W; 107.5 W/cm(2)) both with and without the addition of cytoskeletal-directed agents to assess whether cytoskeletal-directed agents can potentiate ultrasonic sensitivity in different leukemia lines. Human hematopoietic stem cells (hHSCs) and leukocytes were sonicated with continuous 23.5 kHz ultrasound (20 W; 10 W/cm(2)) to determine whether this approach elicited the preferential damage of neoplastic cells over normal blood components. To determine whether ultrasonic sensitivity is exclusively dependent on cell size, leukemia cells were also enlarged via alteration of cell growth parameters including serum deprivation and re-addition, and plateau-phase subculturing. Results indicated that cytochalasin B/ultrasound treatments had the highest rates of initial U937 cell damage. The cells enlarged and partially synchronized, either by serum deprivation and re-addition or by plateau-phase subculturing and synchronous release, were not comparably sensitive to ultrasonic destruction based solely on their cell size. In addition, cytochalasin B significantly potentiated the ultrasonic sensitivity of all neoplastic cell lines, but not in normal blood cells, suggesting that preferential damage is attainable with this treatment protocol. Therefore, it is likely that ultrasonic cell lysis depends not only on cell size and type, but also on the specific molecular mechanisms used to induce cell enlargement and their effects on cell integrity. This is supported by the fact that either the microfilament-or microtubule-disrupting agent produced a higher rate of lysis for cells of a given size than the corresponding stabilizing agents.

N-Farnesyloxy-norcantharimide and N-farnesyl-norcantharimide inhibit the progression of leukemia and increase survival days in a syngeneic mouse leukemia model.

This study investigated the anticancer effects of two newly synthesized norcantharidin analogs, N-farnesyloxy-norcantharimide (NOC15) and N-farnesyl-norcantharimide (NC15), in L1210 cells and in a syngeneic mouse leukemia model (L1210 cell line plus DBA/2 mice). We found that the half-maximal inhibitory concentration (IC50) of NOC15 and NC15 on L1210 cells is 1.56 and 2.62 µmol/l, respectively, and that the IC50 of NOC15 and NC15 on human normal lymphoblast is 207.9 and 2569 µmol/l, respectively. In cell cycle analysis, NOC15 could increase the sub-G1 phase, whereas NC15 could induce G2/M arrest. Annexin-V apoptosis assay indicated that both NOC15 and NC15 could induce cell apoptosis. In the syngeneic mouse leukemia model, both NOC15 and NC15 could increase the survival days of mice and decrease the tumor weight. Moreover, both NOC15 and NC15 could retard the increase in peripheral blood leukocyte count due to L1210 cells. In the subcutaneous (s.c.) group, the treatment with NOC15 could retard the decrease in the weight of the liver and the spleen caused by L1210 cells, whereas the treatment with NC15 could retard the decrease in the weight of the spleen caused by L1210 cells. We conclude that the new compounds NOC15 and NC15 have strong anticancer activity and low toxicity both in vitro and in vivo. NOC15 and NC15 may have the potential to be developed into anticancer agents in the future.

Investigation of fatty acid conjugates of 3,5-bisarylmethylene-4-piperidone derivatives as antitumor agents and human topoisomerase-IIα inhibitors.

A series of five 3,5-bisarylidene-4-piperidones designed as analogs of curcumin and their twenty five fatty acid conjugates were synthesized as candidate anticancer agents. The fatty acid conjugates were designed for efficient delivery of these compounds at the targeted cancer sites. The cytostatic potential of these compounds was evaluated against three representative cancer cell lines namely murine leukemic L1210 cells, and human T-lymphocyte CEM cells and cervical HeLa cells. Most compounds were found to exhibit significant anti-cancer activity in vitro. QSAR studies indicated electrophilicity of these compounds towards cellular nucleophiles may have a key role to play in their cytostatic activity. Representative compounds were also tested for topoisomerase IIα inhibitory potential, which indicated strong catalytic inhibition of the enzyme in vitro. The data showed that the fatty acid conjugates also possessed robust antioxidant activity in multiple analyses. This study also indicated that these compounds prompted significantly lower cellular damage in human fibroblasts than a currently used cancer drug sorafenib in vitro. The wide spectrum of anticancer action, supplemented with antioxidant potential along with non-toxic manifestations, certainly augment the anticancer candidacy of the novel fatty acid conjugates.

The synthesis, characterisation and cytotoxicity of bisintercalating (2,2':6',2''-terpyridine)platinum(II) complexes.

Dinuclear (2,2':6',2''-terpyridine)platinum(II) (PtTerpy) complexes were synthesised by tethering either thiol or pyridine based linkers. All intermediates and resulting complexes were characterised using a combination of (1)H and (195)Pt NMR, two-dimensional (1)H correlation spectroscopy (NOSY/COSY), two-dimensional (1)H/(195)Pt heteronuclear multiple bond correlation spectroscopy (HMQC), elemental analysis and electrospray ionisation mass spectrometry (ESI-MS). The cytotoxicity of the complexes was determined against human A2780 ovarian carcinoma cells and its cisplatin-resistant sub-line A2780cis, as well as L1210 murine leukemia cells.

Tyrosine phosphorylation of HSC70 and its interaction with RFC mediates methotrexate resistance in murine L1210 leukemia cells.

We previously identified and characterized a 66-68 kDa membrane-associated, tyrosine phosphorylated protein in murine leukemia L1210 cells as HSC70 which is a methotrexate (MTX)-binding protein. In order to further characterize the functional role of HSC70 in regulating MTX resistance in L1210 cells, we first showed that HSC70 colocalizes and interacts with reduced folate carrier (RFC) in L1210 cells by confocal laser scanning microscopy and Duolink in situ proximity ligation assay. The tyrosine phosphorylation status of HSC70 found in the membrane fraction was different from the parental L1210/0 and cisplatin (CDDP)-MTX cross resistant L1210/DDP cells. In MTX-binding assays, HSC70 from L1210/DDP cells showed less affinity for MTX-agarose beads than that of L1210/0 cells. In addition, genistein (a tyrosine phosphorylation inhibitor) significantly enhanced the resistance of L1210/0 cells to MTX. Moreover, site-directed mutation studies indicated the importance of tyrosine phosphorylation of HSC70 in regulating its binding to MTX. These findings suggest that tyrosine phosphorylation of HSC70 regulates the transportation of MTX into the cells via the HSC70-RFC system and contributes to MTX resistance in L1210 cells.

A novel class of bis- and tris-chelate diam(m)inebis(dicarboxylato)platinum(IV) complexes as potential anticancer prodrugs.

A novel class of platinum(IV) complexes of the type [Pt(Am)(R(COO)2)2], where Am is a chelating diamine or two monodentate am(m)ine ligands and R(COO)2 is a chelating dicarboxylato moiety, was synthesized. For this purpose, the reaction between the corresponding tetrahydroxidoplatinum(IV) precursors and various dicarboxylic acids, such as oxalic, malonic, 3-methylmalonic, and cyclobutanedicarboxylic acid, was utilized. All new compounds were characterized in detail, using 1D and 2D NMR techniques, ESI-MS, FTIR spectroscopy, elemental analysis, TGA, and X-ray diffraction. Their in vitro cytotoxicity was determined in a panel of human tumor cell lines (CH1, SW480 and A549) by means of the MTT colorimetric assay. Furthermore, the lipophilicity and redox properties of the novel complexes were evaluated in order to better understand their pharmacological behavior. The most promising drug candidate, 4b (Pt(DACH)(mal)2), demonstrated low in vivo toxicity but profound anticancer activity against both the L1210 leukemia and CT-26 colon carcinoma models.

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.

Organometallic nucleoside analogues with ferrocenyl linker groups: synthesis and cancer cell line studies.

Examples of organometallic compounds as nucleoside analogues are rare within the field of medicinal bioorganometallic chemistry. We report on the synthesis and properties of two chiral ferrocene derivatives containing a nucleobase and a hydroxyalkyl group. These so-called ferronucleosides show promising anticancer activity, with cytostatic studies on five different cancer cell lines indicating that both functional groups are required for optimal activity.

A new lyngbyatoxin from the Hawaiian cyanobacterium Moorea producens.

Lyngbyatoxin A from the marine cyanobacterium Moorea producens (formerly Lyngbya majuscula) is known as the causative agent of "swimmer's itch" with its highly inflammatory effect. A new toxic compound was isolated along with lyngbyatoxin A from an ethyl acetate extract of M. producens collected from Hawaii. Analyses of HR-ESI-MS and NMR spectroscopies revealed the isolated compound had the same planar structure with that of lyngbyatoxin A. The results of optical rotation and CD spectra indicated that the compound was a new lyngbyatoxin A derivative, 12-epi-lyngbyatoxin A (1). While 12-epi-lyngbyatoxin A showed comparable toxicities with lyngbyatoxin A in cytotoxicity and crustacean lethality tests, it showed more than 100 times lower affinity for protein kinase Cδ (PKCδ) using the PKCδ-C1B peptide when compared to lyngbyatoxin A.

Induction of apoptosis associated with chromosomal DNA fragmentation and caspase-3 activation in leukemia L1210 cells by TiO2 nanoparticles.

We investigated the effects of nanosized TiO2 particles on the death of mouse leukemia L1210 cells. TiO2 particles suppressed proliferation and induced cell death, as measured by lactate dehydrogenase (LDH) release into the culture medium. Chromatin condensation, which is typical of the initiation of cell death, was observed in approximately 14% cells cultured with titanium dioxide (TiO2) particles for 12 h. Furthermore, giant DNA fragments of approximately 2 Mbp and high-molecular-weight DNA fragments between 100 kbp and 1 Mbp were observed in cells cultured for 18 h with TiO2 particles. These giant and high-molecular-weight DNA fragments were further degraded into smaller DNA fragments, appearing as DNA ladders. Corresponding to the generation of DNA fragments, caspase-3 activity increased in cells treated with TiO2 particles. TiO2 particle-induced LDH release was not inhibited by cytochalasin D, an inhibitor of endocytosis. These results suggest that nanosized TiO2 particles can induce apoptosis associated with DNA fragmentation and caspase-3 activation and that TiO2 particle-induced apoptosis is not caused by endocytosis but is associated with contact of the particles with the cell surface.

Pegylated liposomal mitoxantrone is more therapeutically active than mitoxantrone in L1210 ascitic tumor and exhibits dose-dependent activity saturation effect.

Our previous studies have proved that encapsulation of mitoxantrone into pegylated SUVs (plm60-s) could enhance its antineoplastic efficacy (Li et al., 2008b). However, why plm60-s is more therapeutically active than free mitoxantrone (MIT), and whether pharmacokinetics and activity of plm60-s exhibits dose-dependency are left unknown. In studies with L1210 ascitic tumor-bearing mice in which the dose of MIT was elevated from 2 to 8mg/kg, a saturation of antineoplastic efficacy was observed after plm60-s, and not after free MIT therapy. Total MIT concentrations in plasma, liver and ascitic fluids after plm60-s increased linearly with escalated doses. The released MIT concentrations in ascitic fluid increased continuously before reaching the peak at a dose of 6mg/kg and then decreased. In vitro release experiments using ascitic fluid as release medium revealed that at high concentrations of plm60-s the release of drug was inhibited. At a dose of 4mg/kg, the areas under the curve (AUC) of released MIT in ascitic fluid after plm60-s were higher than those after free MIT, which might be responsible for the enhanced efficacy of plm60-s. These observations may be used to choose a dose regimen of plm60-s to ensure optimal efficacy and to expound the reasons why plm60-s was more therapeutically active.

A novel ribonuclease with antiproliferative activity toward leukemia and lymphoma cells and HIV-1 reverse transcriptase inhibitory activity from the mushroom, Hohenbuehelia serotina.

In this study, a 27-kDa ribonuclease (RNase) was purified from the dried fruiting bodies of the mushroom, Hohenbuehelia serotina. The isolation protocol involved anion exchange chromatography, affinity chromatography, cation exchange chromatography and gel filtration in succession. The RNase was unadsorbed on DEAE-cellulose, but was adsorbed on Affi-gel blue gel and CM-cellulose. The N-terminal amino acid sequence was TVGGSLAEKGN which showed homology to other fungal RNases to a certain degree. The RNase exhibited maximal RNase activity at pH 5 and 80˚C. It demonstrated the highest ribonucleolytic activity toward poly(C), a relatively high activity toward poly(U), and a considerably weaker activity toward poly(A) and (G). The RNase inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase with an IC50 of 50 µM and reduced [3H-methyl]-thymidine uptake by L1210 leukemia cells and MBL2 lymphoma cells with an IC50 of 25 µM and 40 µM, respectively.