Proliferation-dependent cytotoxicity of methotrexate in murine L5178Y leukemia.

The basis for the proliferation-dependent cytotoxicity of methotrexate has been investigated in mice bearing the L5178Y ascites leukemia. Methotrexate at 60 mg/kg i.p. reduced the viability of logarithmically growing ascites cells (55% active S phase cells) to 28% of control, whereas the viability of the slowly growing cells (18% active S phase) was decreased to only 59% of control. Log phase tumor cells accumulated 8-fold higher levels of methotrexate polyglutamates compared to cells that had approached the stationary phase. However, no differences between log phase and slowly growing tumor cells were observed in the cellular levels of unmetabolized methotrexate. Intestinal mucosa and bone marrow from non-tumor-bearing mice resembled slowly growing tumor cells and had markedly lower levels of methotrexate polyglutamates than logarithmically growing cells. The greater accumulation of methotrexate polyglutamates in the logarithmically growing tumor cells was consistent with an increased synthesis of methotrexate polyglutamates in these cells. The enhanced methotrexate polyglutamylation in log phase versus slowly growing cells was not related to changes in the rates of either cellular methotrexate transport, transmembrane efflux of methotrexate, or hydrolysis of methotrexate polyglutamates. Thymidylate synthase activity measured in situ and in extracts from log phase cells was 4- and 2-fold higher, respectively, than in the more slowly growing cells. Methotrexate produced a 2.4-fold greater depletion of poly-gamma-glutamyl derivatives of 5,10-methylenetetrahydropteroylglutamate in log phase cells compared to slowly growing cells, and this was a function of both the increased methotrexate polyglutamate accumulation and thymidylate synthase activity in the rapidly proliferating cells. These results provide further evidence that the selectivity of methotrexate for tumors with a high growth fraction is a consequence of the rapid rates of both cellular methotrexate polyglutamate synthesis and oxidation of 5,10-methylenetetrahydropteroyl polyglutamates by thymidylate synthase.

L-asparaginase-induced modulation of methotrexate polyglutamylation in murine leukemia L5178Y.

The modulation of methotrexate polyglutamylation by L-asparaginase has been examined in mice bearing sublines of leukemia L5178Y that have different sensitivities to asparaginase. A single i.p. injection of 200 IU/kg of asparaginase completely inhibited ascites tumor cell growth in the parental L5178Y/S+ tumor for 120 h compared to 72 and 30 h in the L5178Y/S and L5178Y/S+/- sublines, respectively. Similarly, DNA and protein synthesis were completely inhibited by asparaginase for 96 h in L5178Y/S+ cells, but only for 72 and 24 h in L5178Y/S and L5178Y/S+/- cells. In each tumor the temporal patterns of depletion and recovery of S-phase cells were similar to the patterns of suppression and recovery of DNA and protein synthesis observed in that tumor. When methotrexate was administered at either 96 or 24 h after asparaginase during the asparaginase-induced S-phase nadirs of L5178Y/S+ and L5178Y/S+/- cells, respectively, subsequent methotrexate polyglutamylation was inhibited 83 and 92% compared to tumor cells exposed to methotrexate only. Recovery of methotrexate polyglutamylation in both tumors following L-asparaginase pretreatment coincided in time with the return in the fraction of S-phase cells towards the pretreatment values. The inhibition of methotrexate polyglutamate accumulation by asparaginase was associated with decreased retention of methotrexate in tumor cells. In contrast, asparaginase had no significant effect on methotrexate polyglutamate accumulation and methotrexate retention when administered after methotrexate. These data indicated that the asparaginase-induced modulation of methotrexate polyglutamylation in mice was directly related to the time course of inhibition and recovery of tumor cell proliferation by asparaginase, and thus varied with the intrinsic sensitivity of the individual tumor to the enzyme.

5-Nitrofuran derivatives of fatty acid hydrazides induce differentiation in human myeloid leukaemic cell lines.

Compounds formed by 5-nitrofuran with hydrazides of formic, acetic and propionic acids, hereafter respectively known as SBF, SBA and SBP have been used to evaluate the differentiation-inducing properties on two established myeloid leukaemic cell lines ML-2 and EOL-1. SBP is found to be the most effective as an antineoplastic agent amongst the three. Induction of differentiation observed are in the order SBP > SBA > SBF, as assessed by morphology, NBT-reducing activity and surface marker antigens of the treated cells. Induction of differentiation of ML-2 and EOL-1 cells by the most effective compound, SBP (3 microM), is accompanied by perturbation of the cell cycle, with most of the cells accumulating in the G0-G1, phase. Inhibition of DNA synthesis occurs while protein and RNA synthesis remain practically unchanged.

Effects of methotrexate on folates in Krebs ascites and L1210 murine leukemia cells.

Changes in reduced folates upon exposure of Krebs ascites cells and L1210 murine leukemia cells to methotrexate (MTX) have been measured by stoichiometric entrapment of tissue methylenetetrahydrofolate into a stable ternary complex with thymidylate synthase and tritiated 5-fluoro-2'-deoxy-uridine-5'-monophosphate. Tetrahydrofolate and 5-methyltetrahydrofolate were determined after conversion to methylenetetrahydrofolate. In both tumor cell lines, treatment with methotrexate at levels which had little effect on methylenetetrahydrofolate and tetrahydrofolate concentrations resulted in nearly complete elimination of the methyltetrahydrofolate pool. Thus, an initial effect of methotrexate on folate metabolism appears to be on methyltetrahydrofolate.

Antiprotozoal activity of diospyrin towards Leishmania donovani promastigotes in vitro.

Diospyrin, a bis-naphthoquinone derivative, isolated from a plant, known for its antitumour properties against Ehrlich ascites carcinoma in Swiss A mice, exhibits antiprotozoal activity towards L. donovani promastigotes in culture.

Anti-inflammatory activity of tea (Camellia sinensis) root extract.

Pharmacological studies were carried out with methanol-water (1:1) extract of dried tea (Camellia sinensis) root extract (TRE). TRE was found to possess anti-inflammatory, analgesic and antipyretic activities at 1/10th of its LD50 dose of 100 mg/kg i.p. It was found that TRE inhibited the arachidonic acid-induced paw oedema in rats which indicated that TRE produced the anti-inflammatory activity by inhibiting both the cyclooxygenase and lypooxygenase pathways of arachidonic acid metabolism. TRE also enhanced peritoneal cell count and the number of macrophages in normal mice. It is plausible that the saponins present in TRE may be responsible for these activities of TRE.

Determination of mouse liver 5-methyltetrahydrofolate concentration and polyglutamate forms.

The concentration and polyglutamate status of 5-methyltetrahydrofolate in mouse liver tissue extracts has been determined by enzymatic conversion to methylenetetrahydrofolate and subsequent entrapment of this cofactor form into a ternary complex with Lactobacillus casei thymidylate synthase and tritiated 5-fluorodeoxyuridylate. 5-Methyltetrahydrofolate was oxidized to methylenetetrahydrofolate using the reverse reaction of methylenetetrahydrofolate reductase with menadione as the ultimate electron acceptor. Reference 5-methyltetrahydrofolate could be quantitatively recovered from tissue extracts by this method. The polyglutamate status of enzymatically converted and complexed tissue 5-methyltetrahydrofolate was determined electrophoretically. Unlabeled 5-fluorodeoxyuridylate was used to remove endogenous methylenetetrahydrofolate prior to enzymatic oxidation of 5-methyltetrahydrofolate and subsequent electrophoretic analysis. In this manner, the 5-methyltetrahydrofolate polyglutamate pool alone could be labeled and visualized. There were no observable differences in the polyglutamate distribution of endogenous methylenetetrahydrofolate versus 5-methyltetrahydrofolate polyglutamates in extracts of normal mouse liver tissue.

Chloroaceto hydroxamic acid as antitumor agent against Ehrlich ascites carcinoma in mice.

In vivo cell growth inhibition of Ehrlich ascites carcinoma (EAC) has been evaluated with chloroacetohydroxamic acid, (CHA), having -CH2 Cl, for the -NH2 group of hydroxyurea (HU). The inhibitory character of CHA against EAC in mice model has been found to be comparable with that of HU. Cell growth inhibition by CHA is accompanied by inhibitions of DNA and protein synthesis of the treated cells. The transplantability of EAC cells treated with a single dose of (100 mg/kg) CHA is found to be reduced. Enhanced intraperitoneal macrophage is observed in normal mice following CHA (100 mg/kg) treatment. Deviations of hematological parameters and alkaline phosphatase (ALKP) activity consequent to tumor growth are found to be recovered in tumor bearing mice treated with CHA. All these studies suggest the importance of CHA for further trial as a potent antitumor agent.

A fraction isolated from Ehrlich ascites carcinoma as an antitumor and differentiating agent against human leukemic cell ML-2.

Lipopolysaccharide fraction isolated from Ehrlich ascites carcinoma (E-LPS) was investigated as an antitumor agent against human leukemia cell ML-2. Marked cell growth inhibition was observed with ML-2 cell accompanied by inhibition of DNA synthesis and perturbation of cell cycle. Induction of differentiation in treated ML-2 cells was observed as indicated by morphological maturation, NBT reducing activity and indirect immunofluorescence.

Antitumor properties of boron complexes with hydroxy biguanide and salicyl hydroxamic acid against Ehrlich ascites carcinoma.

A new derivative of hydroxamic acid, hydroxy biguanido hydrochloride monohydrate and its boron derivative, dihydroxy-oxybiguanido boron (III) hydrochloride monohydrate were synthesized. Another boron compound, hydroxo-salicyl-hydroxamato boron (III) was synthesized from known salicyl hydroxamic acid. Antitumor properties of all the compounds evaluated against Ehrlich ascites carcinoma in mice show enhanced survival time when boron is incorporated in the compounds. Hematological parameters, alkaline phosphatase in serum of the treated animals show minimum toxic effects after boron is coupled with their respective hydroxamic acids.

On the possible use of a new boron compound, hydroxysalicylhydroxamato boron(III), and ultrasound in the treatment of female mice bearing the Ehrlich ascites carcinoma cells.

The inhibitory effects of a new boron compound, hydroxy salicylhydroxamato boron (III) (SHB) and ultrasound of frequency 25 KHz (US) on the growth of ascites tumor in female Swiss mice were studied by monitoring the survival and the tumor growth in the treated tumor bearing mice and also the transplantability and the DNA synthesis in the treated tumor (Ehrlich ascites carcinoma) cells. While SHB alone produced a highly significant antitumor activity, US alone produced a small but significant effect. The combination of SHB and US produced significantly greater antitumor activity than SHB alone. The mechanisms of SHB and US actionary are discussed.

Boron compounds against human leukemic cells.

Two new boron compoumds, dihydroxy(oxybiguanido) boron (iii) hydrochloride monohydrate (HB) and guanidine biboric acid adduct (GB) were used in this study to observe the antitumor effect. Leukemic blast cells isolated from chronic myeloid leukemia (CML) patients showed significant cell growth inhibition within twentyfour hours. IC50 of GB and HB was 2mg/ml. The metabolically active cells were found to be inhibited by drug treatment as assessed by MTT test. Inhibition of 3H Thymidine incorporation also supported the above result. In this study we investigated the molecular mechanisms by which HB and GB induce apoptosis in immature blast cells.

Antineoplastic effect of new boron compounds against leukemic cell lines and cells from leukemic patients.

Three new boron compounds, dihydroxy (oxybiguanido) boron (iii) hydrochloride monohydrate (HB), guanidine biboric acid adduct (GB) and hydroxosalicyl hydroxomato boron (iii) (SHB) were studied to observe their antineoplastic effect, if any. Leukemic cells isolated from acute lymphatic leukaemia (ALL) patients and chronic myeloid leukaemia patients (CML) and myeloid leukemia cell lines (HL 60 and U-937) showed cell growth inhibition after treatment with the boron compounds. MTT assay showed that the growth of metabolically active cells was inhibited by treatment with these drugs. The molecular mechanism by which SHB induced apoptosis in immature blast cells was also investigated by ladder formation in gel electrophoresis.

Studies with black tea and its constituents on leukemic cells and cell lines.

The anticancer effect of black tea (BT) and its polyphenols theaflavin (TF) and thearubigin (TR) has been evaluated on U-937 cell line, a myeloid leukemic cell line and on leukemic cells isolated from peripheral blood of chronic myeloid leukemia (CML) patients. In both types of cells, cell growth inhibition was observed 24 hrs after treatment with BT, TF and TR. MTT assay showed growth inhibition of metabolically active cells and inhibition of DNA synthesis was observed by 3H-Thymidine incorporation after treatment with the compounds. In all cases TF and TR were more effective than BT, suggesting that these are possibly the active components in BT responsible for its antileukemic activity. Superoxide dismutase (SOD), a free radical scavenger, was found to be increased by TF, whereas BT and TR lowered the level in comparison to the control. The present study is the first report of antileukemic effect of BT and its polyphenols.

An antitumor pectic polysaccharide from Feronia limonia.

An acidic heteropolysaccharide has been isolated from the tropical angiosperm Feronia limonia syn. F. elephantum (family: Rutaceae). A partially carboxymethylated alpha-(1-4) polygalacturonan backbone structure with 2- and 2,4-O-alpha-L-rhamnopyranosyl, 2- and 2, 3-O-alpha-L-arabinofuranosyl and 3-, 2,4-and terminal alpha-D-galactopyranosyl bearing side chains has been tentatively assigned. The preliminary study in the murine model showed some significant in vivo Ehrlich ascites carcinoma cell growth inhibition.

Reversal of methotrexate-induced folate pool depletion by thymidine in a human leukemia cell line in vitro.

In an in vitro study using a human monocytic leukemia cell line, U-937, the effects of interferon-gamma (IFN-gamma) in combination with the antifolate methotrexate and the role of thymidine introduced as a biochemical modulator were investigated. Methotrexate alone or in combination with INF-gamma was found to enhance the induction of morphologic and functional monocytic differentiation in the U-937 cell line. Various cellular effects with the addition of thymidine to the medium with methotrexate and IFN-gamma were studied. Enhanced inhibition of cell growth and perturbation of the cell cycle were noted when methotrexate and IFN-gamma were used in combination, but not when methotrexate was used alone. The reduction of cellular folate by methotrexate was also enhanced in combination with IFN-gamma. Cell cycle delay, resulting in cell growth inhibition of folate depletion, caused the induction of differentiation in U-937 cells, which was found to be greater with methotrexate + IFN-gamma than with methotrexate alone. Cellular differentiation, as assessed by nitroblue tetrazolium reduction assay, indirect immunofluorescence and morphology, showed better effects towards the differentiation of U-937 cells when the agents were used in combination. However, addition of thymidine to the medium was found to cancel all the aforementioned effects. The addition of thymidine to the medium also caused reversal of the inhibitory effect of methotrexate and IFN-gamma on cell growth and repletion of the endogenous folate level. Repletion of the folate level by exogenous thymidine is a new possibility for the role of the thymidine in cellular growth.