Melting of cross-linked DNA IV. Methods for computer modeling of total influence on DNA melting of monofunctional adducts, intrastrand and interstrand cross-links formed by molecules of an antitumor drug.

A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition.

Gallium chloride effects on neonatal rat heart cells in culture, in standard and oxidative conditions.

The effects of gallium chloride (GaCl3) at 7.17, 28.68 and 114.7 microns (0.5, 2 and 8 mg/l of Ga3+) were checked in cardiac cells derived from 2-4 day-old newborn rats, cultured for 72 h in Eagle's minimum essential medium (MEM), enriched with 10% foetal calf serum (v/v) and 2 mM of glutamine at 37 degrees C, with 95% air plus 5% CO2. After 3 hours of standard culture conditions (MEM with glucose 5 mM), Ga treatment induced an increase of glycogen stores without any influence on ATP, ADP, and AMP concentrations. A slight and transient decrease in the beat rate was noted after 15 min of exposure to GaCl3 at all concentrations, whereas there was no difference in the beat rate nor in the cell contraction amplitude after 3 hours of exposure. After 1.5 h in conditions of oxidation (Tyrode solution without glucose, FeCl2 20 microM, ascorbic acid 0.2 mM), GaCl3 at 8 mg/l decreased the malondialdehyde (MDA) production as assessed by the decrease of intracellular concentrations and the decrease of its release in the supernatant. The decreased MDA production following oxidative stress, the increase in glycogen stores in normal oxygen concentrations, as well as the maintenance of ATP concentrations and the lack of any chronotropic effect induced by GaCl3 suggests a protective rather than a deleterious cardiac effect.

Preclinical toxicology and tissue gallium distribution of a novel antitumour gallium compound: tris (8-quinolinolato) gallium (III).

Tris (8-chinolinolato) gallium (III) compound (KP46), a new organo-metallic gallium (Ga) complex, has been synthesized for potential use in anticancer therapy. Although this agent has a better bioavailability after oral administration than Ga chloride, it also shows a greater toxicity. The purpose of the present study was to assess the acute and subacute toxicity of KP46, and to determine tissue Ga distribution in healthy Swiss mice. Ga assays were performed by inductively coupled plasma atomic emission spectrometry. In the first experiment, the drug was given by gavage at single doses ranging from 464 to 4640 mg/kg. The LD50 values were 2870 mg/kg (410 mg Ga3+/kg) and 2370 mg/kg (339 mg Ga3+/kg) for males and females, respectively. In the second experiment, KP46 was administered by gavage at 0, 62.5, 125, 250, 500 and 750 mg/kg/day for two weeks (8 animals/dose/sex). The dose of 62.5 mg/kg/day was well tolerated, without deaths, decreased weight gain, or renal, hepatic and hematological toxicities. Higher doses decrease the probability of survival. A significant decrease in the number of white blood cells was noted at doses of 125 mg/kg/day (p<0.05), while hemoglobin, hepatic and renal functions were not affected. At 62.5 mg/kg/day, the Ga concentrations were 7.02 +/- 3.14 microg/g in bone, 3.55 +/- 2.10 microg/g in the liver, 1.81 +/- 0.24 microg/g in the kidneys, 1.77 +/- 1.45 microg/g in the spleen. In lungs, brain, testes and ovaries, the Ga concentrations were under the limit of detection (0.030 ng/g). According to these results, the therapeutic potential of KP46, orally administered, should be evaluated with the dose of 62.5 mg/kg/day. The great affinity of Ga for bone could justify the consideration of KP46 for malignant bone tumours.

Relationship between magnesium, cancer and carcinogenic or anticancer metals.

It is known that carcinogenesis induces Mg distribution disturbances which cause Mg mobilization through blood cells and Mg depletion in non-neoplastic tissues. Mg deficiency seems to be carcinogenic in cases of some particular haemolymphoreticular diseases only, more often, in cases of solid tumors, it inhibits carcinogenesis. Both carcinogenesis and Mg deficiency increase the plasma membrane permeability and fluidity. The regulation of Mg cellular and subcellular distribution is very important in cancer research. If the Mg level is normal in histones, and particularly in H1 and in nucleic acids on the N7 site of the guanine, it can protect against carcinogenesis. On the other hand, if it is abnormally high it can generate the left-handed Z DNA which is correlated with carcinogenesis. The carcinogenic action of Mg may be linked with some effects on sulfur amino-acid metabolism, growth and the immune system. Although Mg antagonizes certain carcinogenic metals, this is not one of its general properties. Our studies on human amniotic membranes show that Mg acts as a competitive antagonist on 2 or 3 weak carcinogens, Pb and Cd, but not on Co. Mg is a non-competitive antagonist of Ni and is devoid of action on As, both of which are powerful carcinogens. All carcinogenic metals (As, Ni, Cr, Cd, Co, Pb, except Be) reduce the total conductance (Gt). Reversibly all cocarcinogens increase Gt or do not modify it. However, both anticancer and carcinogenic metals reduce Gt. Thus the main target in the relationship between Mg and metals in carcinogenesis is either membrane as regards Pb and Cd, or nucleus as regards Pt, Ga, Co and Ni. The ideal treatment of Mg disturbances in carcinogenesis should control the factors which regulate cellular and subcellular Mg distribution in neoplastic tissues as well as those which avoid Mg depletion in extra malignant tissues.

Effects of gallium chloride oral administration on transplanted C3HBA mammary adenocarcinoma: Ga, Mg, Ca and Fe concentration and anatomopathological characteristics.

To determine the influence of the length of the treatment on the anatomopathological and biochemical intratumor changes induced by gallium, we treated C3H BA mammary adenocarcinoma-bearing C3H/HeJ mice with gallium chloride daily, for a period of either 21 or 42 days. In both cases the same dose of 200 mg/kg/24h was administered. An increase of collagen fibrosis in treated tumors as opposed to controls was only noted after 42 days, as well as a significant decrease of the intratumor magnesium and calcium concentrations that could be responsible for a reduction in the metabolic activities of the malignant cells. Remarkable intratumor gallium concentrations (38.4 +/- 30.3 nmol/g after 21 days of treatment; 13.4 +/- 7.3 nmol/g after 42 days where the necrosis is much more important) are obtained after this oral administration. There is no renal toxicity and a higher tumor/kidney concentration ratio is obtained than after acute parenteral administration. The effect of gallium may be different according to the mode of administration: it may be more cytotoxic after parenteral administration, while after oral administration it may act as a better metabolic regulator with a more selective tumor uptake and fewer side effects.

Acute effects of gallium chloride on ventricular function and metabolism of the Langendorff perfused rat heart.

The effects of two concentrations of GaCl3 (1.79 microM and 7.17 microM) were studied on isolated perfused paced rat hearts. All hearts were submitted to an equilibration period of 20 minutes under normal conditions of oxygenation (95% O2, 5% CO2) and with 11 mM glucose in Krebs-Henseleit buffer. At the end of the perfusion (80 min) tissue Ga contents were 98.0 +/- 13.8 and 200.2 +/- 28.5 nM/g of wet weight for the lower and the higher Ga concentrations respectively. Left ventricular developed pressure (LVdp) as well as +LVdp/dt and -LVdp/dt were similar in control and Ga-treated groups during the 60 minutes following the equilibration period. At the same time mean coronary flow and oxygen consumption were lower (p < 0.05) in hearts perfused with 7.17 microM Ga than in the control group. Lactate production did not differ in the control and Ga-treated groups. Mean creatine kinase release was lower (p < 0.05) in the 7.17 microM Ga-treated group than in the 1.79 microM Ga-treated and control groups. Intratissular malondialdehyde as well as glycogen and ATP concentrations did not differ in all groups at the end of the experiment. Gallium chloride partially prevented the unavoidable oedema resulting from using saline Krebs-Henseleit solution. In conclusion, acute GaCl3 administration improves the functionality of the Langendorff-heart model.

Inhibitory effects of gallium chloride and tris (8-quinolinolato) gallium III on A549 human malignant cell line.

The effects of two gallium (Ga) compounds, Ga chloride (GaCl3) and tris(8-quinolinolato)Ga (III) on the viability of A549 human malignant lung adenocarcinoma cells were investigated. The results demonstrated that both drugs reduced the viability of A549 cells but to different extents. The inhibitory effects of tris(8-quinolinolato)Ga (III) were 10 times more profound than those produced by GaCl3. The IC50 was obtained with 2.5 microM of tris(8-quinolinolato)Ga (III) and 25 microM GaCl3 after an exposure time of 48 hours. Further, whereas the inhibitory effects of GaCl3 were both dose and time-dependent those of tris(8-quinolinolato)Ga (III) appeared to be only dose-dependent, indicating differences in their mechanism of action. Comparison with data drawn from the literature suggests that GaCl3 seems to be in the same range of activity as Ga nitrate or Ga-pyridoxal isocotinoyl hydrazone. Tris(8-quinolinolato)Ga (III) could be as effective as transferrin-Ga, but with the advantage of oral administration and a greater bioavailability of the tris(8-quinolinolato)Ga (III) compound.

Effect of gallium at two phases of the CA 755 tumour growth.

GaCl3 efficiency was evaluated for mice mammary adenocarcinoma Ca-755 separately at two stages of tumour growth: the exponential and plateau phase. Two modalities of drug administration were evaluated: once only and twice a day for five days. Comparisons were made with the efficiency observed when saline liquid and cyclophosphamide were used. The comparisons between tumour shrinkages were carried out by non-parametric tests. GaCl3 was more often efficient for tumours at the exponential phase than for those at the plateau phase of growth. GaCl3 always appeared less efficient than cyclophosphamide. The treatment schedule did not result in any significant alteration of tumour regression.

Combination chemotherapy with cisplatin, etoposide and gallium chloride for lung cancer: individual adaptation of doses.

Twelve inoperable lung cancer patients were treated with a combination chemotherapy of cisplatinum (CDDP) and etoposide (VP16), as a continuous infusion for 5 days, every 21 days, and with a daily oral administration of GaCl3. Dosages of CDDP and VP16 were adapted in order to obtain an area under the curve (AUC) of 80,000 micrograms l-1.h for plasma total platinum and of 200 mumol.l-1 h for plasma VP16 during each 120 h infusion. GaCl3 was given at the dosage of 400 mg/24h from the time of diagnosis at least until the evaluation after 3 courses of chemotherapy. An objective response was observed in 5 non small cell (NSCLC) lung cancer patients (group 1) and 3 small cell (SCLC) lung cancer patients (group 2). In the other 4 patients with a NSCLC no partial response was noted (group 3). No significant difference in area under the curve (AUC) was noted between the 3 groups, either for plasma total platinum (group 1 = 89,598 +/- 20,843 micrograms l-1.h; group 2 = 88,081 +/- 15,431 micrograms l-1.h; group 3 = 83,820 +/- 13,455 micrograms l-1.h), or for VP16 (group 1 = 227 +/- 41 mumol.l-1 h; group 2 = 217 +/- 29 mumol.l-1.h and group 3 = 211 +/- 30 mumol.l-1.h). The maximal plasma Ga concentrations were 244 +/- 34 micrograms/l in group 1, 112 +/- 57 micrograms/l in group 3 (p less than 0.005) and 243 +/- 132 micrograms/l in group 2. It was then decided to increase the dose of GaCl3 in the further non-responding patients. In 6 responders, 3 additional courses of this combination chemotherapy could have been given without major toxicity, allowing a much more important decrease in the tumor volume in 4 of them. This schedule of treatment should permit the chemotherapy to continue for longer than 6 courses, in order to improve the survival time.

Dose optimization of gallium chloride, orally administered, in combination with platinum compounds.

An individual dose adaptation for cisplatin (CDDP), etoposide and gallium chloride (GaCl3) was proposed to improve the efficacy of this combination chemotherapy and avoid its toxicity. A clinical study was performed in 28 non small cell lung cancer patients, to verify this hypothesis. CDDP and etoposide were administered as continuous infusions every 3 weeks and GaCl3 orally during and between the CDDP-etoposide sequential infusions. CDDP doses were adjusted to achieve, during each 5 day infusion, an area under the total plasma platinum concentrations versus time curve (AUC Pt 0-120) ranging between 80,000 and 100,000 micrograms/l.h. Etoposide dosages were 120 mg/24 h during days 1-3 of the CDDP infusion. GaCl3 dosages were adjusted to obtain plasma gallium (Ga) concentrations ranging between 200 and 400 micrograms/l. The proposed methods of adaptation were successful from a pharmacokinetic point of view as AUC Pt 0-120 were respectively 81351 +/- 4788, 88268 +/- 8451 and 88331 +/- 8778 micrograms/l.h during the first 3 courses, and plasma Ga concentrations, determined during the 2nd and 3rd CDDP courses, 16 hours after the beginning of the CDDP infusion, were respectively 264 +/- 127 and 313 +/- 186 micrograms/l. However, these results were not pharmacodynamically successful and the therapeutic window was not confirmed. Past clinical trials with GaCl3 will be reviewed, as well as the factors which modify the pharmacokinetics or the pharmacodynamic effects of CDDP and GaCl3. From this review, an optimal dosage of 400 mg GaCl3 could be proposed to potentiate a combination chemotherapy with a platinum compound. The target AUC of the platinum compound should be the AUC avoiding its cumulative toxicity.

Beneficial effects of a vanadium complex with cysteine, administered at low doses on benzo(alpha)pyrene-induced leiomyosarcomas in Wistar rats.

BACKGROUND: Vanadium is a potent environmental and body metal, possessing remarkable antitumor and antidiabetic properties. Vanadium salts and complexes have been widely investigated for their anticarcinogenic properties in experimental carcinogenesis. In the present study the antitumor effects of a new vanadium complex with cysteine in relation to identical doses of vanadyl sulfate and cysteine, in tumor bearing rats are investigated. MATERIALS AND METHODS: Male wistar rats were injected with benzo(alpha)pyrene and divided into four groups of 21 rats each. Control group was treated only with BaP. The first group(TR-1) was treated by vanadyl sulfate per os at daily doses of 0.5 mg of V/kg b.w per day. The second (TR-2) by cysteine at doses of 4.5 mg/kg b.w per day and the third group (TR-3), by the complex V(III)-cysteine at daily doses of V 0.5 mg/kg b.w (containing cysteine at concentrations of 4.5 mg/b.w). Treatment was started when tumors were developed (evidenced from a palbable mass at the site of Bap injection) and went on till death. Toxicological tests were performed in 27 rats divided into a control group and two test groups; T-1 administered with vanadyl sulfate at daily doses of 18.5 mg V/kg b.w and T-2 group with V(III)-cysteine complex at daily doses of 18.5 V/kg b.w, for 9 weeks. Mean survival time, death rate, tumor growth rate, the carcinogenic potency of BaP, and the anticarcinogenic potency in relation to histological findings in each treatment group were calculated in each group in order to evaluate the antitumor effects of the substances used. RESULTS: Vanadyl sulfate, cysteine and V(III)-cysteine exerted antitumor effects on leiomyosarcoma bearing Wistar rats. However, V(III)-complex exerted much more potent effects than the other treatments, significantly prolonging mean survival time, retarding tumor growth rate and decreasing the carcinogenic potency of BaP in the TR-3 group, in comparison to the control and the TR-1 and TR-2 groups. Moreover V(III)-cysteine complex resulted in complete remission of 4 (19.7%) of the tumor bearing rats. Blood, urine, biochemical routine tests as well as autopsy did not reveal any toxic effects either of vanadyl sulafate or V(III)-cysteine complex. CONCLUSIONS: Vanadyl sulfate, cysteine and V(III)-cysteine complex exerted antitumor effects in tumor bearing rats. The V(III)-cysteine complex, however, exerts much more potent effects, as evident from the results of the present study. These beneficial effects of the above complex, in combination with its low toxicity provide evidence suggest its possible application in the treatment of human malignant diseases.

Clinical pharmacology of gallium chloride after oral administration in lung cancer patients.

Pharmacokinetic parameters were determined in 18 lung cancer patients after a single administration of 800 mg/24 h of GaCl3: Cmax = 123 +/- 61 mu/l; Tmax = 5.2 +/- 5.5 h; AUCO-96h = 4690 +/- 3358 micrograms.l-1.h; AUCO - infinity = 6394 +/- 5352 micrograms.l-1.h; T 1/2 beta = 43 +/- 19 h. Serum Ga concentrations at the steady-state (Css) were then determined in these patients after a daily oral administration of 800 mg/24 h of GaCl3 for 15 days: Css = 274 +/- 167 micrograms/l. No correlation was found between Css and the previous pharmacokinetic parameters in each patient. Various doses of GaCl3 were administered daily to 45 patients to correlate Css and dosage. Serum Ga concentrations increased with dosage from 100 to 400 mg/24 h (p less than 0.05), but not with further dosages up to 1400 mg/24 h. The optimal daily dose of GaCl3 in lung cancer patients seems to be 400 mg/24 h. In 2 patients, Ga was assayed after death in tissues. Ga concentrations were more than 10 micrograms/g in metastases, 3.6 +/- 2.9 micrograms/g in the primary tumor and 2.3 +/- 0.9 micrograms/g in the kidney. Due to the lack of renal and hematological toxicities and the significant uptake of Ga by the tumor, GaCl3 can be used orally in conjunction with other cytotoxic agents. We intend to evaluate its efficacy according to a randomized study comparing chemotherapy versus chemotherapy plus 400 mg/24 h of GaCl3.

Transferrin conjugates of doxorubicin: synthesis, characterization, cellular uptake, and in vitro efficacy.

One strategy for improving the antitumor selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing suitable carrier proteins. Thus, thiolated human serum transferrin was conjugated with four maleimide derivatives of doxorubicin that differed in the stability of the chemical link between drug and spacer. Of the maleimide derivatives, 3-maleimidobenzoic or 4-maleimidophenylacetic acid was bound to the 3'-amino position of doxorubicin through a benzoyl or phenylacetyl amide bond, and 3-maleimidobenzoic acid hydrazide or 4-maleimidophenylacetic acid hydrazide was bound to the 13-keto position through a benzoyl hydrazone or phenylacetyl hydrazone bond. The acid-sensitive transferrin conjugates prepared with the carboxylic hydrazone doxorubicin derivatives exhibited an inhibitory efficacy in the MDA-MB-468 breast cancer cell line and U937 leukemia cell line comparable to that of the free drug (employing the BrdU (5-bromo-2'-deoxyuridine) incorporation assay and tritiated thymidine incorporation assay, respectively, IC50 approximately 0.1-1 mM), whereas conjugates with the amide derivatives showed no activity. Furthermore, antiproliferative activity of the most active transferrin conjugate (i.e. the conjugate containing a benzoyl hydrazone link) was demonstrated in the LXFL 529 lung carcinoma cell line employing a sulforhodamine B assay. In contrast to in vitro studies in tumor cells, cell culture experiments performed with human endothelial cells (HUVEC) showed that the acid-sensitive transferrin conjugates of doxorubicin were significantly less active than free doxorubicin (IC50 values approximately 10-40 higher by the BrdU incorporation assay), indicating selectivity of the doxorubicin-transferrin conjugates for tumor cells. Fluorescence microscopy studies in the MDA-MB-468 breast cancer cell showed that free doxorubicin accumulates in the cell nucleus, whereas doxorubicin of the transferrin conjugates is found localized primarily in the cytoplasm. The differences in the intracellular distribution between transferrin-doxorubicin conjugates and doxorubicin were confirmed by laser scanning confocal microscopy in LXFL 529 cells after a 24 h incubation that revealed an uptake and mode of action other than intercalation with DNA. The relationship between stability, cellular uptake, and cytotoxicity of the conjugates is discussed.

[Associated granular cell tumor and esophageal cancer (author's transl)]. / Association d'une tumeur à cellules granuleuses et d'un cancer de l'oesophage.

The first reported case of an association of Abrikossof's tumor and a cancer in the esophagus is described. After a general view of the published literature on these tumors, their clinical and pathological characteristics are outlined, and their histogenesis envisaged in relation to histochemical and ultrastructural findings. Therapeutic methods and means of follow-up surveillance are defined.

Magnesium and potassium in diabetes and carbohydrate metabolism. Review of the present status and recent results.

Diabetes mellitus is the most common pathological state in which secondary magnesium deficiency occurs. Magnesium metabolism abnormalities vary according to the multiple clinical forms of diabetes: plasma magnesium is more often decreased than red blood cell magnesium. Plasma Mg levels are correlated mainly with the severity of the diabetic state, glucose disposal and endogenous insulin secretion. Various mechanisms are involved in the induction of Mg depletion in diabetes mellitus, i.e. insulin and epinephrine secretion, modifications of the vitamin D metabolism, decrease of blood P, vitamin B6 and taurine levels, increase of vitamin B5, C and glutathione turnover, treatment with high levels of insulin and biguanides. K depletion in diabetes mellitus is well known. Some of its mechanisms are concomitant to those of Mg depletion. But their hierarchic importance is not the same: i.e., insulin hyposecretion is more important versus K+ than versus Mg2+. Insulin increases the cellular inflow of K+ more than that of Mg2+ because there is more free K+ (87%) than Mg2+ (30%) in the cell. The consequences of the double Mg-K depletion are either antagonistic: i.e. versus insulin secretion (increased by K+, decreased by Mg2+) or agonistic i.e. on the membrane: (i.e. Na+K+ATPase), tolerance of glucose oral load, renal disturbances. The real importance of these disorders in the diabetic condition is still poorly understood. Retinopathy and microangiopathy are correlated with the drop of plasma and red blood cell Mg. K deficiency increases the noxious cardiorenal effects of Mg deficiency. The treatment should primarily insure diabetic control.(ABSTRACT TRUNCATED AT 250 WORDS)