Anticancer activity and mechanisms of diacetyldianhydrogalactitol on hepatoma QGY-7703 cells.

Diacetyldianhydrogalactitol (DADAG) is a member of the hexitols which shows a significant anticancer effect. Despite the fact that the antitumor effects of DADAG have been studied in a number of cell lines, the mechanism of its action remains unclear. Herein, we explored antitumor effects of DADAG and the possible mechanisms by which it inhibited the growth of human hepatocellular carcinoma cell QGY-7,703 and its derived xenograft tumors. Cell proliferation was evaluated with the sulforhodamine B assay in vitro. The results suggested that DADAG had mild antiproliferative activity on QGY-7,703 cells. The antitumor effect of DADAG was assessed in nude mice xenografted with QGY-7,703 cells. We found that DADAG significantly inhibited the tumor growth. Flow cytometry results indicated that the retarded cell proliferation is associated with increased G2/M cell cycle arrest. Further studies showed that the induced G2/M cell cycle arrest is, at least partially, attributed to an upregulation of cyclin B1, phospho-cell division cycle 2 (cdc2) (Thr), phospho-cdc2 (Thr), and cdc25c protein expression, and a decrease in cdc2 protein expression. Taken together, our data show that DADAG has mild proliferative effects on QGY-7,703 cells in vitro, but it significantly inhibits the growth of QGY-7,703 in a xenograft model in vivo. The modulation of several cell cycle progression regulation proteins responsible for G2/M phase transition may account for its antitumor effects.

[Apoptosis of hepatocarcinoma cell line HLE induced by the combination of wild type P53 gene and 1,2:5,6-dianhydro-3,4-diacetylgalactitol].

BACKGROUND & OBJECTIVE: P53 gene is a tumor suppressor gene,and its mutation in human tumor cells is frequently observed. Previous studies revealed that wild type p53 (wt-p53)gene can suppress proliferation ,and induce apoptosis of tumor cells. However,the enhancive effect of wt-p53 on apoptosis of tumor cells is not so obvious when it is used alone. Therefore,it is a new field for tumor research that wt-p53 gene combined with drug is used to enhance apoptosis rate of tumor cells. This study was to investigate the enhancement effect of the combination of wt-p53 and 1,2:5,6-dianhydro-3,4-diacetylgalactitol (DADAG)on apoptosis of human hepatocarcinoma cell line HLE. METHODS: HLE cells were transfected with pUHD10-3-p53 plasmid contained wt-p53 gene,and treated with DADAG. After 96-hour treatment,apoptosis was evaluated by flow cytometry and DNA electrophoresis. RESULTS: The apoptosis rates were: 1.4% in untreated group, 3.5% in pUHD10-3-transfection group, 32.6% in DADAG group, 43.4% in pUHD10-3-p53-transfection group, and 74.6% in pUHD10-3-p53-transfection plus DADAG-treatment group. DNA ladder was observed in pUHD10-3-p53-transfection plus DADAG-treatment group. CONCLUSION: Apoptosis of HLE cells could be induced by both wt-p53 gene and DADAG,and the effect was more obvious when HLE cells were treated by the combination of wt-p53 gene and DADAG.

Caspases promoted DADAG-induced apoptosis in human leukemia HL-60 cells.

AIM: To investigate the roles of caspases in diacetyldianhydrogalactitol (DADAG)-induced apoptosis in human leukemia HL-60 cells. METHODS: Inhibition of proliferation was measured by MTT assay. DADAG-induced apoptosis in HL-60 cells was observed by electron microscopy, flow cytometry, and DNA fragmentation assay. Caspase-3 activity was determined by ApoAlert CPP32 colorimetric assay kit. The cleavage of substrates of caspases was detected by Western blot. RESULTS: DADAG exhibited potent antiproliferative activity and induced apoptosis in HL-60 cells. After treatment with DADAG 8 mg/L for 24 h, caspase-3 activity increased markedly and the cleavage of poly-(ADP-ribose) polymerase (PARP), lamin B, and DFF45 appeared. All of the apoptotic signals were suppressed by z-VAD fmk (a general inhibitor of caspase activities), whereas z-DEVD fmk, a selective inhibitor of caspase-3 activity, only induced partial reversion of the apoptotic effects. CONCLUSION: DADAG induced apoptosis in HL-60 cells by activating caspases. Caspases promoted apoptosis through the cleavage of substrates of PARP, lamin B, and DFF45.

[Apoptosis induced by diacetyldianhydrogalactitol and its mechanism in HL-60 leukemia cells].

AIM: To investigate the apoptosis induced by diacetyldianhydrogalactitol (DADAG) and its mechanism in human HL-60 leukemia cells. METHODS: Inhibition of proliferation was measured by MTT assay. DADAG-induced apoptosis in HL-60 cells was observed by electron microscopy, flow cytometry and DNA fragmentation assay. The levels of Bcl-2 family proteins were detected by Western blotting. Caspase-3 activity was determined by ApoAlert CPP32 colorimetric assay kit. RESULTS: DADAG exhibited potent antiproliferative activity and induced apoptosis in HL-60 cells. After treatment with DADAG 8 micrograms.mL-1 for various times, the Bcl-XL protein level decreased in a time-dependent manner, while the Bad protein level was upregulated. The caspase-3 activity increased markedly after treatment with DADAG for 24 h. The apoptotic signals were suppressed by z-VAD.fmk (a general inhibitor of caspases), whereas z-DEVD.fmk, a selective inhibitor of caspase-3, only induced partial reversion of the apoptotic effects. CONCLUSION: DADAG-induced apoptosis in HL-60 cells required caspase-3 activation and caspase-3 activation was related with Bcl-2 family members.

Combined treatment of anaplastic astrocytoma (grade 3-4) with diacetyl-dianhydro-galactitol (DADAG).

Autoradiographic studies of labeled diacetyldianhydro-galactitol (DADAG) with tumor bearing animals revealed that the CNS accumulates high amounts of DADAG-derived radioactivity and the elimination from the brain seems to be relatively slow. This observation and the activity of DADAG against murine ependymoblastoma classified the drug as a promising agent for the treatment of malignant brain tumors. In a series of 30 evaluable consecutive patients who were operated on for anaplastic astrocytomas, DADAG has been applied during and subsequent to postoperative radiotherapy. No severe toxicity occurred. Survivals were compared with a group of patients who got irradiation alone. Statistical analysis did not show significantly better survivals in the DADAG treated group: median value was 46.5 weeks, p = 0.232.

[The effect of 3,4-disuccinyldianhydrogalactitol, N-nitrosourea and their combination on DNA synthesis in normal and mouse P388 tumor cells]. / Vliianie 3,4-disuktsinildiangidrogalaktitola, N-nitrozomochevin i ikh kombinatsii na sintez DNK v normal'nykh i opukholevykh kletkakh myshei s leikozom P388.

The rates of incorporation of 2-14C-thymidine into DNA of leukemia P388, bone marrow, gastrointestinal mucosa and spleen cells at various time after administration of 3,4-disuccinyldianhydrogalactitol (DisuDAG), 1-methyl-1-nitrosourea (MNU), 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea (HECNU) and their combinations at different doses to mice with leukemia P388 (solid form) were studied. DisuDAG (80 mg/kg) induced the deep and the stable inhibition in DNA synthesis of leukemia P388, bone marrow and spleen cells. The combination of DisuDAG and HECNU at small doses induced the deep and the stable suppression of DNA synthesis in tumor cells, however DNA synthesis in normal dividing cells was shown to recover more rapidly than in leukemia P388 cells. Administration of the combination of DisuDAG with MNU to tumor-bearing mice induced more stable inhibition of DNA synthesis in tumor cells in comparison with MNU and DisuDAG. In vivo inhibition of DNA synthesis in leukemia P388 cells with DisuDAG and HECNU was not due to damage in pool of precursors (TCA soluble fraction).

Absence of cross-resistance between two alkylating agents: BCNU vs bifunctional galactitol.

Dianhydrogalactitol (DAG) increased the life span of both BCNU-sensitive and -resistant L1210 tumor-bearing mice. However, the BCNU-resistant strain showed slightly lower sensitivity against DAG, which could be overcome by an increase in drug dose of ca. 20%. The somewhat lower sensitivity was proportional to a slightly reduced DNA cross-linking formation induced by DAG in BCNU-resistant cells. The amount of DNA cross-links was determined by measurement of the 1,6-di(guaninyl)-galactitol content of DNA. The slight reduction in cross-links is not attributable to DNA repair but rather to other factors that seem to prevent the formation of DNA-drug adducts. The absence of cross-resistance is explained by different kinds of DNA damage caused by the two alkylating agents and the presumably different defense mechanisms developed by cells against these lesions.

The role of comparative pharmacokinetics in the planning of human dose escalation: the experience with diacetyldianhydrogalactitol.

The pharmacokinetics of diacetyldianhydrogalactitol (DADAG) was compared in mice, rats, and humans. The ratios of human therapeutic dose (ThD) to the LD10 were 8 and 5 in mice and rats, respectively. The ratios of the corresponding AUCs of DADAG were 20 and 17, whereas those of dianhydrogalactitol (DAG), the main, active metabolite of DADAG, were 8 in both species. The lower human-to-rodent ratio for DAG was due to the fact that twice as much DAG was formed in the animals. Other factors contributing to the larger AUC in man were the 3-5 times smaller distribution volume found in humans as well as the lower hexitol sensitivity of human bone marrow cells. We conclude that in addition to the distance between the AUCs of the LD10 and of the human starting dose, interspecies pharmacokinetic differences should also be considered in planning the rate of dose escalation.

Development and some characteristics of a P388 leukemia strain resistant to 1, 2:5, 6-dianhydrogalactitol.

Repeated intraperitoneal treatment of standard P388 mouse leukemia with dianhydrogalactitol (DAG) resulted in the development of a P388/DAG experimental mouse tumor which was resistant to the drug. Resistance was stable without DAG treatment throughout 80 passages. P388/DAG shows cross-resistance to alkylating agents such as nitrogen-mustard, cyclophosphamide and diacetyl-DAG but not to selected antimetabolites and tubulin binders and exhibits reduced sensitivity to nitrosoureas. Resistance to DAG could not be overcome by the administration of maximally tolerated dose of DAG to tumor bearing mice. The resistant tumor is one chromosome short and shows a 13-fold increase of cells possessing a submetacentric marker chromosome.

Pharmacokinetic study in a phase I trial with an alkylating agent, diacetyldianhydrogalactitol (DADAG).

Diacetyldianhydrogalactitol (DADAG), a new alkylating hexitol derivative, was given in 30-min infusions for 5 consecutive days or as a single high-dose administration. The parent drug was eliminated in a biphasic manner, with a terminal half-life of 30-40 h. Dianhydrogalactitol (DAG), the main, pharmacologically active metabolite, appeared after a lag time of about 0.2-0.6 h. Its peak concentration was reached 1-2 h after termination of the infusion. The terminal elimination of DAG followed that of the parent compound. During the 5-day schedule slight accumulation was observed, and the plasma concentrations of both compounds approached the steady state. Over a dose range of 75-1050 mg/m2 the daily mean plasma concentrations of DADAG increased by only about 3-4 times. Dose-dependent expansions of the distribution volumes of the drug (Vc, V lambda, Vss) were observed. The behavior of DADAG and DAG in the body could be adequately described by a three-compartment open model. After equilibration the plasma levels of the parent compound and its metabolite were determined by the rate of return of DADAG from the peripheral compartment and its conversion to DAG.

Relation between dose, plasma concentration and toxicity in a phase I trial using high dose intermittent administration of an alkylating agent, diacetyldianhydrogalactitol (DADAG).

Diacetyldianhydrogalactitol (DADAG), a new alkylating sugar alcohol derivative, was administered as single, 30-min infusions in doses ranging from 390 to 1200 mg/m2. The dose-limiting toxicity was myelosuppression. The median times to WBC nadir and regeneration were 16 and 21 days, and to platelet nadir and recovery 20 and 27, respectively. Nausea and vomiting occurred frequently and were of moderate severity. For phase II studies 900 mg/m2 DADAG given every 4-6 weeks is recommended. The area under the plasma concentration time curve (AUC) for DADAG did not increase in proportion with dose escalation; it changed only from 235.5 +/- 70.7 to 262.4 +/- 71.5 micrograms h ml-1 between doses of 690 and 1050 mg/m2. No correlations between the dose administered and the nadir values for haemoglobin concentration, WBC and platelet counts, or the number of episodes of vomiting were demonstrable in this dose range. Such an association was revealed, however, when the above biological variables were related to the individual AUC for DADAG.

Enzymological and morphological changes in rat intestinal mucosa following treatment with alkylating sugar alcohol derivatives.

Wistar rats were treated with alkylating sugar alcohol derivatives, dianhydrogalactitol (DAG) and diacetyldyanhydrogalactitol (Diac-DAG), respectively. The drugs were intravenously administered as a single, bolus injection. The applied doses 2.5, 5, 10, 17 mg/kg DAG and 5, 10, 20, 40 mg/kg Diac-DAG were roughly equitoxic. The effect of these cytostatic agents was studied on the different marker enzymes (thymidine kinase, xanthine oxidase, alkaline phosphatase, sucrase, maltase) of the separated mucosa cells derived from the functional and proliferating zone of the small intestine. Both DAG and Diac-DAG inhibited the enzyme activities of the proliferating and mature enterocytes in a dose dependent fashion, primarily acting on the crypt specific thymidine kinase. The time dependent sequence in the biochemical alterations correlated well with the cytomorphological changes. The drug-induced damage was most pronounced 48 hours after a single treatment. The regeneration of the intestinal mucosa began on days 3 and 4 and was completed by day 7. Diac-DAG at equimolar concentration proved to be more toxic than DAG on the intestine as judged by the significantly higher decrease of protein content and xanthine oxidase activity.

Biochemical changes of intestinal epithelial cells induced by cytostatic agents in rats.

In the intestinal epithelium the rapidly proliferating crypt cells, the precursors of the mature enterocytes are extremely sensitive to the effects of cytostatic agents. The symptoms of intestinal impairment: nausea, vomiting, diarrhea, ulceration, are well known both in clinical practice and in experimental chemotherapy. To obtain information about the biochemical nature of these side effects, a study was performed by investigating the influence of clinically used alkylating hexitol derivatives, dianhydrogalactitol and diacetyl-dianhydrogalactitol, on rat intestinal mucosa cells. The biochemical parameters were investigated in isolated intestinal mucosa cells. Cell proliferation was characterized by measuring the activity of thymidine kinase, while digestion was evaluated by assaying the alkaline phosphatase, sucrase and maltase activities localized in the brush border membrane of the villus cells. The dose response studies of the different enzyme activities indicated that inhibition in all cases was dose dependent. The nadir of the intestinal damage and the time of regeneration were influenced both by the dose and the dosage schedule of the drugs.

[Effect of 1,2:5,6-dianhydrogalactitol and 1,2:5,6-dianhydro-3,4-diacetylgalactitol on DNA synthesis in the cells of melanoma B16, bone marrow, small intestine epithelium, spleen and liver of mice]. / Vliianie 1,2:5,6-diangidrogalaktitola i 1,2:5,6-diangidro-3,4-diatsetilgalaktitola na sintez DNK v kletkakh melanomy B16, kostnogo mozga, épiteliia tonkogo kishechnika. selezenki i pecheni myshei.

Alterations in DNA synthesis induced by 1,2:5,6-dianhydrogalactitol (DAG) and 1,2:5,6-dianhydro-3,4-diacetyldianhydrocalactitol (Diac-DAG) were studied in host normal cells and tumor cells. After administration of these antitumor drugs to melanoma B16-bearing mice, DNA synthesis in host tissues (bone marrow, gastrointestinal mucosa, spleen, liver) got recovered more rapidly than DNA synthesis in melanoma B16. Diac-DAG differed from DAG from the standpoint of damage to DNA synthesis in normal cells. Only DAG inhibited the DNA synthesis in liver cells. Inhibition of DNA synthesis in the bone marrow and spleen with Diac-DAG was less remarkable than with DAG.

Steroid receptors and response of ovarian cancer to cytostatic drugs in vitro.

Samples of 21 ovarian cancers were assayed for oestrogen receptor (ER) and progesterone receptor (PR) content, and the response in vitro to treatment with a combination of doxorubicin, diacetyldian hydrogalactitol and cisplatin was determined. The number of living cells after drug exposure was estimated by a new ATP-bioluminescence method and the tumours were considered responsive if cell survival was less than or equal to 50% of the value in a corresponding control culture. Of the 16 tumours that responded to drug exposure, nine were ER-positive, seven ER-negative and eight were PR-positive, eight PR-negative. The mean percentages of surviving cells ranged from 22.2% in PR-negative tumours to 30.9% in PR-positive tumours. There were no differences in the response rates or in the degree of response to the cytostatics in terms of either receptor status or tumour histology. The results were also compared with those obtained in the same tumour samples exposed to hormones, tamoxifen and medroxyprogesterone acetate. The average response of all tumours was better to cytostatics than to hormones (P less than 0.05); this was particularly marked in the ER-negative tumours. Cytostatics may be preferable to hormones as the primary drug treatment for ovarian cancers but steroid-receptor determinations appear not to help in formulating the optimum drug treatment.

Damages of DNA synthesis in normal and tumor cells with sugar alcohol derivatives.

The rates of incorporation of 2-14C-thymidine into DNA of melanoma B16, bone marrow, gastrointestinal mucosa, spleen and liver at various time after administration of dianhydrogalactitol (DAG), 3,4-diacetyldianhydrogalactitol (DiacDAG) and 3,4-disuccinyldianhydrogalactitol (DisuDAG) at maxima nonlethal single doses to tumor-bearing mice were studied. The sugar alcohol derivatives induced the stable inhibition in DNA synthesis of tumor cells. DNA synthesis in normal dividing cells was shown to recover more rapidly than in melanoma B16 cells after administration of all drugs. DisuDAG is characterized by stronger inhibitory effect on DNA synthesis in melanoma B16 cells at the half of the single maxima nonlethal dose compared with DAG and DiacDAG. Differing from DAG, DiacDAG and DisuDAG did not effect the incorporation of 2-14C-thymidine into DNA of liver cells. In vivo inhibition of DNA synthesis in melanoma B16 cells with DiacDAG was not due to damage of the TCA soluble fraction.

Complex evaluation of the effect of some cytostatic hexitol derivatives.

The antitumor effect of three structurally closely related alkylating hexitol derivatives (DBD, DAG, DiacDAG) was evaluated using a complex multi-parameter evaluation system. It comprised toxicology, action on ascites and solid tumors, as well as on subpopulations isolated by isopyknic centrifugation, cross-resistance and their action on chromatin components (DNA, histone, nonhistone proteins). The results indicate that in spite of their same mode of action, considerable differences could be observed in tumor specificity, inhibition of tumor growth and in their interaction with chromatin components. This multiparameter system seems to be very useful for comparative studies of other alkylating agents, especially for the evaluation of the effect of chemically closely related compounds.