Marcella Coronnello

Melatonin's growth-inhibitory effect on hepatoma AH 130 in the rat

We tested the effects of daily melatonin treatment on the growth of the ascites hepatoma in rats, determining survival time, cell number and cell cycle phases at various stages of tumor development. Melatonin inhibited cellular proliferation, doubled mean life-time and increased survival. Thymidine incorporation in hepatoma cells from treated rats decreased significantly without changes in the apoptotic index. Flow cytometric analysis showed that melatonin slowed cell cycle progression by increasing the number of cells in phase G0G1. Thus, similar to in vitro models, melatonins oncostatic action in vivo appears to be directed to specific cell cycle mechanisms, which remain to be elucidated.

Cytotoxicity, DNA damage, and cell cycle perturbations induced by two representative gold(III) complexes in human leukemic cells with different cisplatin sensitivity.

The gold(III) complexes [Au(phen)Cl2]Cl and [Au(dien)Cl]Cl2 were recently shown to exert important cytotoxic effects in vitro on human tumor cell lines. To elucidate the biochemical mechanisms leading to cell death, the effects produced by these gold(III) complexes on the leukemic CCRF-CEM cell line--either sensitive (CCRF-CEM) or resistant to cisplatin (CCRF-CEM/CDDP)--were analyzed in detail by various techniques. For comparison purposes the effects produced by equitoxic concentrations of cisplatin were also analyzed. First, the dependence of the IC50 values of either complex on the incubation time was investigated. Cytotoxicity experiments confirmed that both gold(III) compounds retain their efficacy against the cisplatin-resistant line: only minimal cross-resistance with cisplatin was detected. Notably, [Au(phen)Cl2]Cl is more cytotoxic than [Au(dien)Cl]Cl2, with IC50 values of 7.4 and 6.0 M at 24 and 72 h, respectively, on the resistant line. Results of the COMET assay point out that both gold(III) complexes directly damage nuclear DNA. Remarkably, DNA damage inferred by either gold(III) complex in the two cell lines is larger than that produced by equitoxic cisplatin concentrations. Finally, the effects that either gold(III) complex produces on the cell cycle were investigated by flow cytometry. It was found that both complexes cause only moderate and transient cell cycle perturbations. Larger cell cycle perturbations are induced by equitoxic concentrations of cisplatin. The implications of the present results for the mechanism of action of cytotoxic gold(III) complexes are discussed.

N,N-bis(cyclohexanol)amine aryl esters: a new class of highly potent transporter-dependent multidrug resistance inhibitors.

A new series of Pgp-dependent MDR inhibitors having a N,N-bis(cyclohexanol)amine scaffold was designed on the basis of the frozen analogue approach. The scaffold chosen gives origin to different geometrical isomers. The new compounds showed a wide range of potencies and efficacies on doxorubicin-resistant erythroleukemia K562 cells in the pirarubicin uptake assay. The most interesting compounds (isomers of 3) were studied further evaluating their action on the ATPase activity present in rat small intestine membrane vesicles and doxorubicin cytotoxicity potentiation on K562 cells. The latter assay was performed also on the isomers of 4. The four isomers of each set present different behavior in each of these tests. Compound 3d shows the most promising properties as it was able to completely reverse Pgp-dependent pirarubicin extrusion at low nanomolar concentration, inhibited ATPase activity at 5 x 10(-9) and increased the cytotoxicity of doxorubicin with a reversal fold (RF) of 36.4 at 3 microM concentration.

Synthesis of new pyrazolo[5,1-c][1,2,4] benzotriazines, pyrazolo[5,1-c]pyrido[4,3-e][1,2,4] triazines and their open analogues as cytotoxic agents in normoxic and hypoxic conditions.

The synthesis and antitumor activity in normoxic and hypoxic conditions of a series of pyrazolo[5,1-c][1,2,4]benzotriazine and its related analogues are reported. All compounds were tested on human colorectal adenocarcinoma cell line HCT-8 and for compounds 15 and 20, which show to have selective cytotoxicity in hypoxic and in normoxic conditions respectively, ROS production, cell cycle, and DNA fragmentation were measured. This preliminary study encouraged us to consider 15 and 20 as interesting leads for further optimization.

Inhibitory effect of luteinising hormone-releasing hormone analogues on human endometrial cancer in vitro

We studied the effects of luteinising hormone-releasing hormone (LHRH) agonist leuproreline (1 microM for 96 h) and LHRH antagonist cetrorelix on the cell growth of primary cultures from nine human endometrial cancers using the sulphorhodamine colorimetric test. Histological examinations and reverse transcription and polymerase chain reaction amplification (RT-PCR) for LHRH receptors were also performed. The endometrial cancers examined had a medium to high degree of proliferative activity and a low degree of apoptotic power; furthermore, they expressed the LHRH receptor RNA variably, detectable in 71% of cases. The addition of leuproreline or cetrorelix to cell cultures inhibited growth in a statistically significant way compared to untreated control cells; nevertheless, the percentage of cell growth inhibition obtained was very variable. These data suggest that LHRH analogues can exert differential inhibitory effects on the growth of endometrial cancer, which seems to be independent of the expression of specific LHRH receptors.

Structure-Activity Relationships Studies in a Series of N,N-Bis(alkanol)amine Aryl Esters as P-Glycoprotein (Pgp) Dependent Multidrug Resistance (MDR) Inhibitors

As a continuation of a previous research, a series of N,N-bis(alkanol)amine aryl esters, as Pgp-dependent MDR inhibitors, was designed and synthesized. The aromatic ester portions are suitably modulated, and new aryl rings (Ar(1) and Ar(2)) were combined with trans-3-(3,4,5-trimethoxyphenyl)vinyl, 3,4,5-trimethoxybenzyl and anthracene moieties that were present in the most potent previously studied compounds. The new compounds showed a wide range of potencies and efficacies on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay. Selected compounds (5, 6, 8, 9, and 21) were further studied, evaluating their action on doxorubicin cytotoxicity potentiation on K562 cells; they significantly enhanced doxorubicin cytotoxicity on K562/DOX cells, confirming the results obtained with pirarubicin. Compound 9 shows the most promising properties as it was able to nearly completely reverse Pgp-dependent pirarubicin extrusion at nanomolar doses and increased the cytotoxicity of doxorubicin with a reversal fold (RF) of 19.1 at 3 microM dose.

Strategy to provide a useful solution to effective delivery of dihydroartemisinin: development, characterization and in vitro studies of liposomal formulations

Dihydroartemisinin is one of the most potent anticancer artemisinin-like compounds, able to induce cancer cell death by apoptotic pathways. Besides its effectiveness, it is a poorly water soluble drug with low bioavailability and low half-life (34-90 min), therefore, the development of new formulations of dihydroartemisinin to increase bioavailability is in great need. Conventional (P90G and cholesterol) and stealth liposomes (P90G; cholesterol and PE 18:0/18:0 PEG 2000) to deliver dihydroartemisinin to cancer cells were developed for the first time. Both developed formulations show physical characteristics as drug carrier for parental administration and good values of encapsulation efficiency (71% conventional liposomes and 69% stealth liposomes). Physical and chemical stabilities were evaluated under storage condition and in presence of albumin. Cellular uptake efficiency of liposomes was determined by flow cytometry. Higher internalization occurred in the conventional liposomes rather than in the stealth liposomes suggesting that hydrophilic steric barrier of PEG molecules can reduce cellular uptake. Flow cytometry analysis was also used as an alternative technique for rapid size determination of liposomes. Cytotoxicity studies in the MCF-7 cell line confirmed the absence of toxicity in blank formulations suggesting liposomes may be a suitable carrier for delivery of DHA avoiding the use of organic solvents. Cytotoxicity of DHA and of both liposomal formulations was evaluated in the same cell line, confirming a modified release of DHA from vesicles after cellular uptake.

Cytotoxic activity of 3-nitropyrazolo[5,1-c][1,2,4]benzotriazine derivatives: a new series of anti-proliferative agents.

We report the synthesis and biological evaluation of a new series of 3-nitropyrazolo[5,1-c][1,2,4]benzotriazine derivatives (compounds 1–4) bearing appropriate substitutions in positions 7 and/or 8. The objective of this investigation was to study the effects of these substitutions on the cytotoxic activity of four new compounds against established human cancer cell lines (i.e. HT29 and HCT-8, colon carcinoma, MCF7, breast carcinoma, and A549, lung carcinoma cells). The inhibitory effects of compounds 1–4 on cell growth were assessed by the sulforhodamine B assay. Also, the effects of these compounds on cell cycle distribution of human colon carcinoma cells (HCT-8) were analyzed by flow cytometry. 3-Nitropyrazolo[5,1-c][1,2,4]benzotriazine derivatives displayed IC50 values in the micromolar range on the growth of the four cell lines tested. Cell cycle perturbations induced on HCT-8 cells by study compounds at the IC50 values consisted prevalently of a slight accumulation of cells in G0/G1 phase and a slight decrease in G2/M phase. However, compound 3 induced a marked accumulation of cells into S phase with concomitant decrease in G0/G1 and G2/M phases. Cytotoxicity data, compared to those obtained with 3-cyano-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (compound 5, NSC 683334) and other compounds previously synthesized in our laboratory, demonstrated a similar or even improved cytotoxic potency. Cell cycle perturbations caused by these compounds support the hypothesis that they may act by a direct or an indirect inhibition of DNA synthesis.

Biochemical modulation of fluoropyrimidines by antifolates and folates in an in vitro model of human leukemia.

Although 5-fluorouracil (FUra) is one of the most effective cytotoxic agents in the treatment of various solid tumors (carcinomas of the gastro-intestinal tract, breast, head and neck), remissions occur in only 20 to 30% of cases and usually are of short duration. Recently, preclinical studies have shown that the antitumor activity of FUra can be potentiated by modulating the metabolism of this drug by using other substances, in particular antifolates of folates. Pretreatment with antifolates may, by blocking de novo purine biosynthesis and consequently increasing phosphoribosyl pyrophosphate (PRPP) pools, enhance the conversion of FUra to active fluoronucleotide pools via orotate phosphoribosyltransferase. Methotrexate (MTX) pretreatment may also enhance binding of the fluoropyrimidine inhibitor, 5-fluodeoxyuridylate (FdUMP), to the target enzyme, thymidylate synthase (TS), indirectly by increasing dihydrofolate polyglutamates or directly, as MTX polyglutamates, by enhancing the formation of ternary complexes with FdUMP and TS. Exogenous folates, in particular 5-formyltetrahydrofolate (folinate, leucovorin, LV), can, by raising the intracellular levels of 5, 10-methylenetetrahydrofolate, lead to increased formation and stabilization of the ternary complex formed by TS, the folate coenzyme, and FdUMP. In vitro studies have also shown potentiation of FUra cytotoxicity by antifolates and folates against human lymphoblastic leukemia cell lines. Thus, while FUra may have little or no single agent activity in leukemias and lymphomas, it may be converted to an active drug in these neoplasms by appropriate modulation. Clinical studies of sequential MTX-FUra or combined LV-FUra based upon experimental tumor results reviewed herein, are warranted.

Multidrug resistance (MDR) reversers: High activity and efficacy in a series of asymmetrical N,N-bis(alkanol)amine aryl esters

As a continuation of our research on potent and efficacious P-gp-dependent multidrug resistance (MDR) reversers, several new N,N-bis(alkanol)amine aryl esters were designed and synthesized, varying the aromatic moieties or the length of the methylenic chain. The new compounds were tested on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay, where most of the new compounds were shown to be active. In particular the asymmetrical compounds, characterized by two linkers of different length, generally showed fairly high activities as MDR reversers. Some selected compounds (isomers 15-17) were further studied by evaluating their doxorubicin cytotoxicity enhancement (reversal fold, RF) on the K562/DOX cell line. The results of both pharmacological assays indicate that compounds 16 (GDE6) and 17 (GDE19) could be interesting leads for the development of new P-gp dependent MDR modulators.