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Abstract Antimutagenic, anticlastogenic, and bioprotective effect of polysaccharide glucomannan (GM) isolated fromCandida utilis was evaluated in four model test systems. The antimutagenic effect of GM against 9-aminoacridine (9-AA)- and sodium azide (NaN3)-induced mutagenicity was revealed in theSalmonella typhimurium strains TA97 and TA100, respectively. GM showed anticlastogenic effect against N-nitroso-N'-methylurea (NMU) induced chromosome aberrations in theVicia sativa assay. The bioprotective effect of GM co-treated with methyl-methane-sulphonate (MMS) was also established inChlamydomonas reinhardtii repair deficient strainsuvs10 anduvs14. The statistically significant antimutagenic potential of GM was not proved against 4-nitro-quinoline-1-oxide (4-NQO)-induced mutagenicity inSaccharomyces cerevisiae D7 assay. It may be due to bioprotectivity of a-mannan and -glucan, which are integral part ofS. cerevisiae cell walls. Due to the good water solubility, low molecular weight (30 kDa), antimutagenic/anticlastogenic, and bioprotective activity against chemical compounds differing in mode of action, GM appears to be a promising natural protective (antimutagenic) agent. [ABSTRACT FROM AUTHOR]

The cytotoxic and antineoplastic potential of two new duplex drugs, ECyd-5-FdU and ECyd- lipid- 5-FdU, were compared with the activity of the parent single-nucleoside analogues, 3-C-ethynylcytidine (ECyd) and 5-fluorodeoxyuridine (5-FdU), either applied as monotherapy or simultaneously in equimolar concentrations simulating their ratio in a duplex drug. Murine leukaemia L1210 cells were used for comparative in vitro tests of the duplex and the single drugs. The tested substances were evaluated for their cytotoxicity, combinatory potential and revitalisation properties. Additionally, an in vivo model of leukaemia L1210-bearing mice of the DBA/2J strain was used for testing of acute toxicity and antileukaemic activity using various chemotherapeutic regimes. Based on the results of this study, the suitability of ECyd and 5-FdU for forming a duplex drug was discussed from the perspective of their expected synergistic anticancer activities. We found an improvement of chemotherapy outcomes of the new duplex drugs tested by comparing their in vitro cytotoxicity and an increase of the time of survival of experimental leukaemia-bearing mice in a statistically significant manner.

The presented review article deals with various conjugates of arabinosylcytosine (araC). This powerful drug that is routinely used in therapy of hematological malignancies has some shortcomings, which limit its use and therapeutic effects. These are low lipophilicity, low stability to degrading enzymes and need for biological activation through phosphorylation. Conjugating araC to another molecule is done with the intention of increasing araC stability and lipophilicity and possibly avoiding rate-limiting araC phosphorylation. An attachment of that another molecule, possessing its own biological activity, may result in formation of a conjugated molecule with new biological activities and better therapeutic potential. The review deals with various araC conjugates formed at the positions N(4), 2, 2', 3' and 5'. Biological activities and differences from araC of compounds formed by conjugation are also discussed.

Cytotoxic/cytostatic activity of N-salicylidene-L-glutamato diaqua copper(II) complex (CuC) against mice leukemia cells L1210 has been estimated and their bioactivity was enhanced by addition of ascorbic acid. The Cu-complex with isoquinoline ligand (IQ-CuC) had stronger cytostatic effect (IC50 =15.6 microM) than parental complex (CuC) and its cytotoxicity several times increased in the presence of 0.1 mM ascorbic acid (IC50 =1.0 microM). The cytotoxicity has been caused by oxidative stress, enhanced creation of TBARS has been confirmed, and formation of 2',7'-dichlorofluorescein from 2',7'- dichlorodihydrofluorescein has been observed, also. Some hallmarks of apoptotic/necrotic death of L1210 cells have been observed by fluorescent microscopy after dyeing of cell with propidium iodide and Hoechst 33342. In addition, it was confirmed that both complexes in the presence of ascorbic acid cleavaged of pDNA. Although these copper complexes were initially prepared as substances with antioxidant properties we have showed that combined treatment of L1210 cells with IQCuC and ascorbic acid induced strong oxidative stress and death of cells. Our results confirmed that physiological concentration of ascorbic acid increases the cytostatic/cytotoxic efficiency of N-salicylidene-L-glutamato diaqua copper(II) complexes.

This review deals with alpha-lipoic acid (LA) from the point of its chemical and biological characteristics affecting enzymatic activities that are part of cellular biochemical processes in normal and cancer cells. This includes attributes of LA that are related to its ability to act as a free-radicals scavenger and also as a radical generator. LA is discussed in the light of its physico-chemical features, toxicity, biochemical bases of LA biological activities, and mechanisms of action. Additionally, it is discussed how these properties of LA are reflected by results of in vivo experiments with cancer cells and in experimental cancer chemotherapy. Finally, the results of LA use in human cancer chemotherapy and as chemopreventive agent are discussed in the light of LA future inclusion into chemotherapeutic protocols.

Polysaccharides represent the major part of the yeast cell wall dry weight and build the skeletal carcass defining cell wall stability and cell morphology (beta-D-glucans) or constitute amorphous matrix and cell surface fibrous material (mannans and mannoproteins). It is known that yeast cell wall beta-D-glucans reveal immunomodulating properties, which allows for their application in anti-infective and antitumor therapy. Recent data also suggest that polysaccharides reveal antioxidant activity that can result in their protective function as antioxidants, antimutagens, and antigenotoxic agents. The paper provides a review of our continuing research involving water-soluble derivatives of beta-D-glucan isolated from the baker's yeast Saccharomyces serevisiae and of a glucomannan isolated from the industrial yeast Candida utilis. The results are confronted with the available literature data. The derivatives of beta-D-glucan demonstrated potent inhibitory effect on lipid peroxidation comparable to that of the known antioxidants and exerted DNA protection from oxidative damage. The free radical scavenging activity was confirmed by spin-trap electron paramagnetic resonance. Antimutagenic and antigenotoxic activity of the yeast polysaccharides was demonstrated using yeast, bacterial, and algal models. The derivatives of beta-D-glucan exerted potent enhancement of tumor necrosis factor alpha (TNF-alpha) released from murine macrophages and revealed synergistic effect with cyclophosphamide in the treatment of Lewis lung carcinoma and two types of lymphosarcoma in murine models. The results indicate significant protective antioxidant, antimutagenic, and antigenotoxic activities of the yeast polysaccharides and imply their potential application in anticancer prevention/therapy.

Various amphiphilic heterodinucleoside phosphates containing 1-beta-D-arabinofuranosylcytosine (ara-C) and 5- fluorodeoxyuridine (5-FdUrd) have recently been synthesized in order to increase the efficacy of ara-C and 5-FdUrd. Employing growth inhibition and growth recovery assays, we evaluated the in vitro effects of four of these dimers (No. 2, 2A, 3, 10) in L1210 and P388D1 murine leukemia cells. Although ara-C and 5-FdUrd appeared equimolar in all dimers, their contribution to the cytotoxicity of these agents was different. Thus, the liberation of ara-C and 5-FdUrd from their dimeric origin and their subsequent metabolic activation had a different course. In another set of experiments, we examined the in vivo effects of these agents in mice. The dimer with the highest cytotoxicity in vitro exerted the lowest acute toxicity and yielded the lowest therapeutic effect in vivo. The obtained data indicate that dimers with slower liberation of ara-C and 5-FdUrd were less cytotoxic, but prolonged liberation of both antimetabolites protected them from inactivation and extended the time period of therapeutic action. Some of the dimers exceeded the synergistic effects yielded by simultaneous application of both ara-C and 5-FdUrd. The significantly higher therapeutic potential of these new antitumor agents indicates that further studies are warranted.

Aim: Cytosine arabinoside is routinely used for treatment of leukemias and lymphomas. However, because of its extensive metabolic inactivation and limited activity in chemotherapy, new analogues of araC are being tested. The aim of this work was to synthetize two araC conjugates and evaluates their cytotoxic/antileukemic activity., Methods: Synthesis of araC-sulfonamide conjugates A and B was performed in anhydrous conditions using cyclostyling and 5'-chlorocyclocytidine as starting material. Elemental analysis and NMR, IR and UV spectrometry were used for structure confirmation. The synthesized araC conjugates were tested for their cytotoxicity in L1210 leukemia cells in vitro and for therapeutic activity and toxicity in vivo in leukemia L1210-bearing mice., Results: The cytotoxic activities of araC and two synthesized conjugates A and B were expressed as IC(50) (micromol/l) and were compared respectively. The conjugate A is 303-times less active and the conjugate B is 757-times less active than araC. Consequently, the antileukemic activity and the acute toxicity of these compounds were examined in experiments involving leukemia L1210-bearing mice. Statistically significant therapeutic outcome was observed when the dosage of both araC conjugates was increased 10-times compared to araC. Next, the ration of cytotoxicity vs therapeutic activity for araC and both conjugates was performed. It was recorded that the ration between cytotoxicity and therapeutic activity for araC is 3333, for the conjugate A and B, the ration is significantly lower (110 and 44). This indicates that the inactivation of araC conjugate A is 30-times slower and the inactivation of conjugate B is 75-times slower as araC inactivation., Conclusions: The differences in cytotoxic and therapeutic activity registered in araC treatment and between two araC-analogues are most probably caused by slow liberation of araC from both conjugates. We are considered that prolonged araC liberation protected them from inactivation and extended the time period of therapeutic action both araC conjugates. The obtained results can serve as stimuli for further investigation of new araC-analogues.

Objectives: The purpose of this study was to determine whether the extract isolated from the artichoke Cynara cardunculus L. (ECC) had antimutagenic effect and was able to enhance the therapeutic effect of cytostatic drug cis-platinum (cis-Pt)., Methods: The potential antimutagenic activity of ECC was assayed by a test on sex-linked recessive lethal mutations detection in Drosophila melanogaster males treated with ethylmethane sulfonate (EMS). The possible enhancement of cytostatic/cytotoxic effect of cis-Pt by ECC was evaluated in the cell revitalization assay by measuring cell viability via Trypan blue exclusive assay using mouse leukemia cells L1210., Results: EMS was both toxic and genotoxic in D. melanogaster males. It statistically significantly increased the frequency of sex-linked recessive lethal mutations in comparison to the negative control. Furthermore, ECC statistically significantly reduced the genotoxic effect of EMS. It acted in a desmutagenic manner via EMS inactivation. In the cell revitalization assay, ECC enhanced the cytotoxic/cytostatic effect of cis-Pt. The therapeutic potential of ECC was established on the basis of statistically significantly lowered recovery of cis-Pt pre-treated mouse leukemia cells in the presence of ECC., Conclusions: The results imply that the extract isolated from artichoke C. cardunculus L. has marked beneficial activities antimutagenic and therapeutic effect enhancing) and its potential biomedical application in the combination therapy of cancer and some neurodegenerative diseases may be suggested.

We describe the use of the new ribonucleotide reductase inhibitor, trimidox (TDX), in combination chemotherapy under in vitro and in vivo conditions with cisplatin and cyclophosphamide. In vitro, the combination of TDX and cisplatin was tested in L1210 cells. The combination caused concentration dependent antagonistic or additive effects. However, the combination of TDX-cisplatin-cyclophosphamide in vivo is highly synergistic in both, the L1210 and P388D1 leukemia mouse models. Both combinations, TDX with cisplatin or TDX with cyclophosphamide were also synergistic in the L1210 and P388D1 leukemia animal models.

Naturally occurring polysaccharides isolated from the yeasts are the substances with versatile intriguing biomodulatory activities. One of the novel derivatives prepared from the (1 --> 3)-beta-D-glucan isolated from the cell walls of baker's yeast Saccharomyces cerevisiae is sulfoethyl glucan (SEG). Its DNA-protective, antimutagenic, anticlastogenic and cytotoxic/cytostatic enhancing effect was evaluated using five eukaryotic systems. SEG showed bioprotective effect in recombination- repair-deficient strain of alga Chlamydomonas reinhardtii against methyl methanesulfonate-induced genotoxicity, antimutagenic effect against ofloxacin-induced genetic changes in yeast Saccharomyces cerevisiae assay and anticlastogenic activity in plants Vicia sativa and Vicia faba assays against maleic hydrazide-induced clastogenicity. In the combined application with cytostatic drug vumon, SEG exerted enhancement of the drug's cytotoxic/cytostatic effect in the cell revitalization assay using mouse leukemia cells. The study sheds light on the possible mechanisms of actions and utilization of this microbial polysaccharide derivative in the cancer prevention and therapy.

Naturally occurring dietary compound resveratrol (RES), possessing chemopreventive and cytostatic properties, has been shown as potent sensitizer for apoptosis induced by a variety of anticancer drugs. Cell cycle analysis in sensitive promyelocytic leukemia HL60 cell line and its multidrug-resistant variant HL60/VCR (P-gp positive) treated with RES resulted in cell cycle arrest in S-phase in both cell variants. Flow cytometry measurements showed diverse activities of RES in combination with anticancer drugs doxorubicin (DOX), cycloheximide (CHX), busulfan (BUS), gemcitabine (GEM) and paclitaxel (PTX), in some cases resulting in apoptosis induction, preferentially at the expense of S-phase. Thus, RES could become a candidate to enhance the efficacy of combination anticancer therapy in a variety of human cancer cells inclusive leukemias.

Nowadays naturally occurring compounds with the potential antimutagenic and anticarcinogenic effects are of great importance for their prospective use in cancer chemoprevention and treatment. The new water soluble derivative of microbial polysaccharide beta-D-glucan-carboxymethyl glucan (CMG) belongs to such a category of natural substances. CMG isolated from the cell wall of baker's yeast Saccharomyces cerevisiae is included into the class of biopolymers known as biological response modifiers (BRMs) with a broad range of activities, above all ones interfering with cancer therapy. It was demonstrated on four experimental model systems that biological and consequential medicinal importance of CMG is based on the combined application with another active compound. In the Saccharomyces cerevisiae antimutagenicity assay CMG significantly reduced ofloxacin-induced mutagenicity in the yeast strain D7. CMG exerted bioprotective (anti-toxic and antimutagenic) effect after its simultaneos application with methyl methanesulphonate on the repair-deficient strain uvs10 of the unicellular green alga Chlamydomonas reinhardtii. In the Vicia sativa simultaneous phytotoxicity and anticlastogenicity assay CMG exerted statistically significant anticlastogenic efect against maleic hydrazide-induced clastogenicity in Vicia sativa L. Only in the Salmonella/microsome assay CMG did not exert statistically significant antigenotoxic effect, despite of the fact that it reduced 9-aminoacridine-induced mutagenicity in S. typhimurium TA97, but his(+) revertants decreasing was statistically significant only at the highest CMG concentration used. The data presented unambiguously documented that even biopolysaccharides (e.g., derivatives of beta-glucan) belonging to the most abundant class of natural biopolymers may contribute to cancer prevention and therapy.

Various heterodinucleoside phosphates of 5-fluorodeoxyuridine (5-FdUrd) and arabinofuranosylcytosine (Ara-C) have recently been synthesized as potent chemotherapeutic agents. 5-Fluorodeoxyuridine is being used in patients with colorectal carcinoma, whereas Ara-C is one of the most effective agents in the treatment of hematological malignancies. We now investigated the action of three novel amphiphilic dimers with different structures in various 5-fluorouracil (5-FU) sensitive and resistant human colon tumor cell lines (CCL228, CCL227, 5-FU resistant CCL227 and HT-29) as well as in L1210 murine leukemia cells. Activity of the heterodimers was determined by clonogenic and growth inhibition assays including the induction of programmed cell death. In addition, the in vivo effects were tested in L1210 leukemia bearing mice. We show that these compounds inhibited the number of colonies of 5-FU sensitive and resistant human colon tumor cell lines with IC50 values ranging from 0.65 to 1 nM. The investigated dimers induced dose-dependent apoptosis in HT-29 colon tumor cells as well as in L1210 leukemia cells. No significant difference in the cytotoxicity of these agents could be observed between 5-FU sensitive and resistant cells, indicating that these compounds might be used in the treatment of 5-FU resistant tumors. In L1210 leukemia bearing mice the survival of tumor-bearing animals was significantly increased in comparison with untreated control animals. We therefore conclude that these new heterodinucleoside phosphates of 5-FdUrd and Ara-C might be an additional option for the treatment of sensitive and 5-FU resistant colon cancer and hematological malignancies.

In this study we have examined the antitumor effect of combined administrations of indomethacin (IND) with doxorubicin (DOX) on growth of K-562 leukemia cells. Although, as single drug treatment, only high concentrations of IND reduced growth (>200 microM) and induced apoptosis (>800 microM) of the K-562 cells, a synergistic effects on DOX-induced cell growth inhibition, apoptosis and differentiation were observed during the co-administration of DOX with 10 microM IND. Cells treated with this combination had elevated GSHt level compare to DOX-treated cells. Modulation of GSHt level of DOX-treated cells with Cd2+ ions or BSO confirmed its important role in processes of DOX-induced differentiation. Results of this study showed that IND has a positive effect on therapeutic efficacy of DOX, and could be a perspective modulator in cancer chemotherapy.

Background: Ara-C (1-beta-D-arabinofuranosylcytosine) is widely used for treatment of human leukemia. However, due to emergence of resistance, new drug combinations need to be developed., Materials and Methods: We tested the combination of Ara-C with 5-FdUrd (5-fluorodeoxyuridine) in L1210 and P388D1 mouse leukemia cells in vitro and in vivo by growth inhibition and recovery assay in leukemia-bearing mice., Results: Simultaneous incubation of cells with Ara-C and 5-FdUrd yielded more than additive effects for most combinations in both cell lines; synergy was seen in P388D1 cells. P388D1 cells showed delayed growth after treatment with Ara-C or 5-FdUrd in a growth recovery assay. In animal studies, simultaneous subcutaneous administration of the compounds did not show any significant beneficial effects as compared with monotherapy. Intraperitoneal administration of both compounds, however, significantly prolonged the survival of P388D1 animals., Conclusion: Depending on cell type and route of drug administration, the combination of Ara-C and 5-FdUrd might offer a promising additional treatment option for leukemia.

9-Bromo-5-morpholino-tetrazolo[1,5-c]quinazoline (BMTQ) acted cytotoxically on murine leukemia cell line L1210 and human colon carcinoma cells Caco-2. We found the two highest concentrations of BMTQ (149.2 and 74.6 microM) induced an acute cytotoxic effect, however other tested concentrations (<74.6 microM) manifested a concentration/dependent and time/dependent cytotoxic effect. The sensitivity of murine leukemia cells L1210 and human colon carcinoma cells Caco-2 was expressed in the same order. The cytotoxicity of BMTQ was not accompanied by changes of the cell cycle profile. Following the cytotoxicity-related effects of BMTQ we observed the induction of ssDNA breaks after BMTQ treatment. All the concentrations of BMTQ increased the level of ssDNA breaks 1.3-2.9 times (after 2 h of treatment) and 1.6-2.8 times (after 4 h of treatment) in Caco-2 cells compared to the control. No apoptotic DNA fragmentation induced by BMTQ in Caco-2 cells was recorded.

Effects of three flavonoids, quercetin (QU), galangin (GA), and chrysin (ChR) on cisplatin (cis-Pt)-induced apoptosis of human promyelocytic leukemia HL-60 cells and murine leukemia L1210 cells were investigated. The quantitative analysis of apoptotic DNA fragmentation was used to show that preincubation of cells with flavonoids can influence cis-Pt-induced apoptosis in different way. ChR had no effect, QU enhanced, and GA reduced apoptotic DNA fragmentation. It is also shown that combined treatment with QU and cis-Pt showed synergistic effect, however, GA combined with cis-Pt exhibited antagonism on cytotoxicity in L1210 murine leukemia cells. We assume that tested flavonoids affect the important biological activities connected with cancer chemotherapy and chemoprevention as they differently modulated the sensitivity of cells to cis-Pt treatment. QU is presented as pro-apoptotic agent and GA as agent with anti-apoptotic potential.

Several metal complex agents have already been introduced into clinical tumor therapy and others are subject of antitumor studies. In this study we focused on the tetraaza macrocyclic copper complex (Cu(TAAB)Cl(2)). We studied the influence of the substance on cell growth, cell cycle, membrane integrity, necrosis, apotosis and glutathione level on the leukemic cell line L1210 in 1-day (22 h) and 3-day (72 h) experiments. The metal complex shows a dose-dependent antiproliferative effect, without affecting cell cycle phases. The present results confirm that copper complex can damage plasmatic membranes and trigger apoptosis, and that after treatment of leukemic cells with the copper complex, glutathione levels were increased.

The stability of the new antileukemic kojic acid derivative, 5-benzyloxy-2-thiocyanatomethyl-4-pyranone (BTMP) was investigated. The degradation of BTMP was studied using specific and reproducible HPLC and LC-MS methods. Accelerated stability studies of BTMP were conducted in 0.1 M hydrochloric acid solution, physiological phosphate buffer solution (pH 7.5) and basic phosphate buffer solution (pH 9.0) at 30, 40 and 60 degrees C, respectively. The degradation of BTMP was found to follow pseudo-first order kinetics. In basic solution (pH 9.0) BTMP underwent rapid hydrolysis at a degradation rate constant (0.183-0.638 h-1) and degradation half-life (3.67-1.06 h) depending on the temperature setting. On the other hand, BTMP was significantly stable in 0.1 M hydrochloric acid solution (kdeg: 0.0017-0.0052 h-1; degradation half-life t1/2: 408.6-135.7 h), whereas in physiological phosphate buffer solution (pH 7.5), BTMP was only moderately stable (kdeg: 0.006-0.231 h-1; degradation half-life: 117.7-3.0 h). Arrhenius plots were constructed to predict the degradation kinetic parameters of BTMP at 25 degrees C and 4 degrees C. LC-MS analyses confirmed the degradation of BTMP in basic solutions and indicated at least two degradation products; namely 5-benzyloxypyran-2-ol-4-one (m/z 217.8) and 2-thiocyanatomethylpyran-5-ol-4-one (m/z 181.6).

Fourteen substituted 4-anilinoquinazolines have been tested for cytotoxic effect and structure activity relationships. The most active derivatives were substituted by chlorine or bromine group in the aromatic ring, in the pyrimidine ring by morpholine group and in the aniline skeleton by nitro group in position 4 or 2. Derivatives 6-bromo-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline, 6-bromo-2-morpholin-1-yl)-4-anilinoquinazoline, 2-(morpholin-1-yl)-4-(4'-bromoanilino)-quinazoline and 6-chloro-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline inhibited growth of tumor cell lines HeLa, B16 and L1210. Mutagenic data provided by Ames test showed, that the compounds 6-bromo-2-morpholin-1-yl)-4-anilinoquinazoline and 2-(morpholin-1-yl)- 4-(4'-bromoanilino)quinazoline did not exhibit the mutagenic effect, whereas the compounds 6-bromo-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline and 6-chloro-2-(morpholin-1-yl)-4-(4'-nitroanilino) quinazoline increased slightly the number of revertants of the strain TA 98 without metabolic activation. Concentration 26 micromol/L of 6-bromo-2-(morpholin-1-yl)-4-anilinoquinazoline induced necrosis of tumor cells B16. Concentration 5.2 micromol/l induced a significant increase of filamentous actin in the transformed HepG2 cells. Derivatives 6-bromo-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline, 6-bromo-2-morpholin-1-yl)-4-anilinoquinazoline, 2-(morpholin-1-yl)-4-(4'-bromoanilino)quinazoline and 6-chloro-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline exhibited antiprotease effect on plasmine. This results could be relevant for the anticancer properties of these compounds.

A DNA protective capacity of three flavonoids, apigenin (AP), luteolin (LU) and quercetin (QU) against free radicals generated by H202, resp. Fe2+ is reported. This effect corresponding with scavenging of free radicals or with chelating of iron was assayed at two concentrations of flavonoids studied (1 microM and 10 microM). The quantitative analysis has shown that LU possesses the highest DNA protective effect of flavonoids investigated in the presence of H2O2. On the other hand, in the presence of 10 microM Fe2+, AP exhibited the highest DNA protective effect at the concentration of 1 microM and the following order was reached at the stoichiometric concentrations (10 microM) of Fe2+. It is believed that this discrepancy is caused by the ability of LU and QU iron-complex formation as it was separately investigated using UV-VIS spectrometry.

Tamoxifen belongs among relatively new drugs. As it has already been shown, it undoubtedly brings a benefit to oncology patients. However, there are still questions regarding its broader use in therapy or cancer prevention. This review puts together some data available at present time with the aim of elucidating the most important aspects of its use in medical oncology.

Kojic acid as a molecule of natural origin may serve as template for the synthesis of new biologically active compounds. The synthetic KA (pyranone) derivatives possess various kinds of biological activities which are related by their similarity to flavonoids. The most important property is the antifungal and antineoplastic activity and capability of chelating metals. It is shown that the antineoplastic activity of kojic acid derivatives is based on various mechanisms of action on different levels of cellular metabolism and functions what makes this compound interesting for future investigation as cytotoxic agent.

5'-Chloro-5'-deoxy arabinosylcytosine (Cl-araC) is a more lipophilic analog of the clinically used drug--arabinosylcytosine (araC). The resistance toward the enzyme cytidine-deaminase action was described as an characteristic feature of this synthetic nucleoside. The kinetics of the Cl-araC transformation in acid and alkaline solutions was studied at various temperatures. When compared with parent compound araC, the chlorine atom at the 5' position of the nucleoside sugar moiety increases the Cl-araC stability. The chlorine atom stabilizing effect is higher in acidic conditions. Cl-araC increased stability, antileukemic activity accompanied by higher lipophilicity confirm the fact that Cl-araC belong among interesting compounds from the point of view of cancer chemotherapy.

Our experiments indicate that administration of a toxic drug with high rate of free-radical formation (doxorubicin, DOX) combined with an antioxidant (alpha-lipoic acid, LA) may lead to a decrease in drug-toxicity. However, the effects of antioxidant may be concentration-dependent and it is therefore crucial to choose its appropriate dosage. LA at a low concentration (1 micromol/l) acts as a growth factor and at a higher concentration (100 micromol/l) acts as an antiproliferation agent. Both concentrations of LA in combination with DOX were examined in cytotoxic and antitumor effects in L1210 mouse leukemia cells employing a MTT chemosensitivity assay. In most concentration combinations, DOX and LA effect were antagonistic and synergistic action was only found at the higher concentration of both agents (DOX 2.5 micromol/l and LA 100 micromol/l). Use of LA in doxorubicin therapy lead to an increase (though marginally significant) in survival of animals. Combined single-dose administration of DOX (5 mg/kg) and LA (16 mg/kg) lead to super-additive effect of the combination on survival of leukemic mice.

Camptothecin (CAM) and cisplatin (cis-diamminedichloroplatinum(II), cis-Pt) were used as inducers of apoptosis in the mouse leukemic L1210 cells. Relatively high concentrations of 50 micromol cis-Pt and 50 micromol CAM, respectively, were used to induce the apoptotic DNA ladder. The simultaneous treatment of L1210 cells by the drug and pentoxifylline (PTX) resulted in a decrease of drug concentrations necessary for the induction of apoptosis. This study revealed that a cell cycle G2 checkpoint inhibitor PTX reduces time intervals necessary for the onset of drug-induced apoptosis in these cells. This fact might be important as the earlier onset of programmed cell death may decrease a risk of tumor cells to become resistant to drug therapy.

The DNA-protective activity of hydroxyurea (HU) and novel ribonucleotide reductase (RR) inhibitors amidox (AX), didox (DX) and trimidox (TX) was examined using hydrogen peroxide as the DNA-damaging agent. The exposure of superspiralized plasmid DNA molecules (pBR 322) to H2O2 under precisely defined in vitro conditions initiates a change in DNA topology (DNA from I relaxes to DNA form II). This electrophoretically monitored change in the plasmid DNA topology is related to the induction of ss-DNA breaks and corresponds with DNA exposition to free radicals. The inhibition of DNA relaxation (the prevention of DNA damage induced by hydrogen peroxide) depended on the free radical scavenging capacity of the drugs investigated. HU exerted DNA protective activity at a concentration of 4 mM, AX at concentration of 1 microM, TX at a concentration of 5 microM and DX at a concentration of 25 microM (the free radical scavenging activity increases from HU to AX in following manner: HU << DX < TX < AX). It can be concluded that the new synthetic RR-inhibitor AX which is being investigated at the preclinical level as a potential anti-cancer drug possess the highest capacity for scavenging of free radicals.

The human pre-B acute lymphoblastic leukemia cell line REH6 was utilized for characterization of CD45 glycoprotein by monoclonal antibodies (mAb) recognizing four distinct CD45 antigen specificities, i.e. nonrestricted CD45, restricted CD45RA, CD45RB and CD45R0. Immunoprecipitation revealed two antigen specificities on REH6 cells of m.w. 220 kDa and 190 kDa, both presenting wide range of isoelectric point pI approximately 6.0-7.5. Nonrestricted CD45 epitopes were not affected by the sialyl acid cleavage with sodium metaperiodate or neuraminidase, but were sensitive to both, tunicamycin, the N-glycosylation inhibitor and monensin, an inhibitor of protein transport through the Golgi compartment. O-sialoglycoprotein endopeptidase from Pasteurella haemolytica A1 partially cleaved CD45RA and CD45RB epitopes, while nonrestricted CD45 determinants were not affected by this enzyme. Limited proteolysis of this antigen resulted in the appearance of 160-180 kDa peptide domains which retained CD45 epitopes. Further, the treatment of cells with phorbol myristate acetate (PMA) induced marked down-regulation of 220 and 190 kDa isoforms and the appearance of new 210, 180 and 170 kDa variant glycoprotein forms which were not found on parental cells. This PMA effect was not accompanied by the programmed cell death and was markedly blocked by a nonselective protein kinase (PK) inhibitor isoquinoline sulfonamide H7. Modulation of CD45 by phorbol esters might serve as an in vitro model for an additional insight into the function of CD45 in hematopoietic cells.

Hydrochloride of 5'-chloro-5'-deoxy-cyclocytidine (Cl-cC) is an analogue of cyclocytine hydrochloride (cC), a prodrug of the compound with the strong antileukemic activity arabinosylcytosine (araC). This paper is devoted to the study of its cytotoxic activity in vitro and to the effect of acid and alkaline conditions and temperature on its stability. Cl-cC inhibits not only the growth of L1210 leukemia cells in vitro and the DNA synthesis (IC50 = 0.09 mumol/l) but, at the same time, it has a weak effect on RNA synthesis (IC50 > 250 mumol/l) and no effect on proteosynthesis. In alkaline conditions Cl-cC is transformed to 5'-chloro-araC and 2',5'-anhydro-araC but is more stable in acid solutions.

1. Occurrence of lesions induced in plasmid DNA by cis-DDP and by HA was quantified both as a transforming activity and as conformation integrity of supercoiled pBR322 DNA. Fifty per cent decrease of the biological activity of plasmid DNA, not accompanied by measurable change of DNA conformation, was observed after a single exposure of DNA to cis-DDP (1 hr/37 degrees C). 2. HA induced conversion of supercoiled DNA to other topological forms in a dose-dependent manner. 3. One- and two-strand DNA breaks were determined electrophoretically with high sensitivity. Cis-DDP exposed DNA relaxed at 30 times lower HA concentration compared to intact DNA. 4. This effect may be connected with a local distortion of DNA structure at the cis-DDP--DNA bond, which makes possible high effectivity of HA-DNA interaction. 5. On other hand, biological activity stayed at the 50% level despite breaks induced in DNA. 6. This finding supports the idea that DNA breaks occur at the locations which were modified during the exposure of DNA to cis-DDP. 7. The importance of the DNA structure during interaction with HA may be seen during HA-DNA interaction at heat-denaturation of supercoiled DNA. At this condition, the DNA breaks were induced at 100 times lower concentration of HA. 8. We conclude, on the basis of these results published earlier, that local distortion of supercoiled DNA structure, which is caused by the cis-DDP bond, and the local DNA uncoiling caused by heat-denaturation are related to high HA-DNA reactivity.

Pyridoxal and cis-diamminedichloroplatinum (II) (cis-DDP) have a different mode of interaction with DNA. Cis-DDP caused extensive transforming inactivation of pBR322 DNA but did not form strand breaks in DNA molecules. On the other hand, pyridoxal formed frank strand breaks in the DNA chains and decreased DNA transforming activity. A higher level of DNA transforming inactivation was obtained during simultaneous application of both compounds than would be an additive effect of these compounds applied to DNA independently. This synergistic effect can be ascribed to the introduction of thermolabile sites by the combined action of cis-DDP and pyridoxal. These lesions were converted by heat-treatment to DNA strand breaks and detected electrophoretically. Using two different methods, cis-DDP and pyridoxal, during simultaneous application, interacted with DNA with higher efficiency.

1. Cytotoxic synergism of drugs cis-diamminedichloroplatinum(II) (cis-DDP) and arabinosylcytosine (araC) was studied both on the level of interaction with DNA in chemically determined conditions and on leukemia L1210 bearing mice. 2. AraC and its structural natural precursor cytidine were tested for the modulation of kinetics of bifunctional adducts production induced by cis-DDP in DNA. 3. This process plays the basic role in cytotoxic mechanism and antitumor activity of cis-DDP. 4. No interaction was seen between cis-DDP and araC. Further, presence of araC in reaction mixture had no effect on cis-DDP-DNA interaction. 5. Therefore, cytotoxic synergism does not arise in the araC-cis-DDP-DNA interaction and its origin is different. 6. Finding that cytidine has no synergistic effect on life span of leukemia L1210 bearing mice when administered together with cis-DDP it shows the difference between cytidine and araC. 7. The small structural difference between cytidine and araC is very important for synergism of cytotoxicity.

The use of conformation isomers of pBR322 DNA in the study of interactions of Pt complexes with DNA provided for a good monitoring of induced changes in the structure of DNA by gel electrophoresis. On the basis of characteristic changes in the gel electrophoretic mobility of platinated isomers of pBR322 DNA we detected the presence of Pt-DNA adducts representing both intra- and interstrand bifunctional binding of Pt complexes to DNA. Also, this method made it possible to distinguish between DNA modifications induced by the therapeutically active cis-diamminedichloroplatinum (II) (cis-DDP) alone, and those induced by its therapeutically inactive trans-isomer (trans-DDP). The electrophoretically detected DNA modifications were more effective if the interaction of the Pt complex took place with heat-denatured DNA. This process, as compared to that performed with native DNA, ran 100 times faster.

The decrease of DNA fluorescence caused by an impaired capacity of ethidium bromide to intercalate into the DNA reflected structural changes caused in the DNA molecule by its interaction with platinum complexes. This fall in DNA fluorescence was proportional to the length of exposure of DNA to the platinum complexes, and depended on the environment in which the interaction took place. The therapeutically active cisplatinum (cis-DDP) was more efficient to inhibit fluorescence in a solution of 4 X 10(-3) mol NaCl than its therapeutically inactive trans-isomer (trans-DDP). For comparison, the inhibition of DNA fluorescence was also studied in a solution of 10(-2) mol NaClO4. The inhibitory effect was elicited more rapidly, but no difference was found between the two isomers. We concluded that the larger effect of cis-DDP on DNA was induced by the 4 X 10(-3) mol concentration of NaCl. Since also the intracellular concentration of chloride ions is 4 X 10(-3) mol, it cannot be ruled out that the interaction between DNA and cis-DDP and trans-DDP in vivo might be influenced by the intracellular environment.