Anna Bielawska

Inhibition of collagen and DNA biosynthesis by a novel amidine analogue of chlorambucil is accompanied by deregulation of β1-integrin and IGF-I receptor signaling in MDA-MB 231 cells

A novel amidine analogue of chlorambucil N-(2-(4-(4-bis(2-chloroethyl)aminophenyl)butyryl)aminoethyl)-5-(4-amidinophenyl)-2-furanecarboxamide hydrochloride (AB(1)), and the parent drug were compared for their effects on collagen and DNA synthesis in breast cancer MDA-MB 231 cells. IC(50) values for chlorambucil and its amidine analogue for collagen synthesis were found to be about 44 and 19μM, respectively. Increased ability of AB(1) to suppress the protein synthesis, compared to chlorambucil, was found to be related to an inhibition of prolidase activity and expression. The phenomena were probably a result of disruption of β(1)-integrin and the insulin-like growth factor-I (IGF-I) receptor mediated signaling caused by this compound. Expression of β(1)-integrin receptor, as well as focal adhesion kinase pp125(FAK) (FAK), growth-factor receptor-bound protein 2 (GRB2), son of sevenless protein 1 (Sos1) and phosphorylated mitogen activated protein kinases (MAPK), extracellular-signal-regulated kinase 1 (ERK(1)) and kinase 2 (ERK(2)) but not Src and Shc proteins was significantly decreased in cells incubated for 24h with 10μM AB(1), compared to controls. Chlorambucil in the same conditions did not evoke any changes in expression of all these signaling proteins, as shown by Western immunoblot analysis. In addition, AB(1) revealed a higher antiproliferative activity than chlorambucil, accompanied by a stronger down-regulation of IGF-I receptor expression. The results were confirmed by [(3)H]thymidine incorporation assay. Incubation of the cells with 10μM AB(1) for 12 and 24h contributed to a decrease in DNA synthesis by about 33 and 46% of the control values, respectively, while in case of chlorambucil by about 23 and 29%, respectively. These data suggest that the amidine analogue of chlorambucil (AB(1)) disturbs MDA-MB 231 cell metabolism more potently than does the parent drug, chlorambucil. The mechanism of this phenomenon may be due to its stronger suppression of β(1)-integrin and IGF-I receptor signaling.

Apoptosis-mediated cytotoxicity of ouabain, digoxin and proscillaridin A in the estrogen independent MDA-MB-231 breast cancer cells

We examined the effects of three cardiac glycosides, ouabain, digoxin and proscillaridin A, on the proliferation of estrogen independent MDA-MB-231 breast cancer cells. In terms of reduction in cell viability, the compounds rank for both 24 h and 48 h of incubation in MDA-MB-231 cells in the order proscillaridin A > digoxin > ouabain. Digoxin for 24 h and 48 h of incubation in MDA-MB-231 cells proved to be only slightly more potent than ouabain, with IC50 values of 122 ± 2 and 70 ± 2 nM, respectively, compared to 150 ± 2 and 90 ± 2 nM for ouabain. In contrast, proscillaridin A, was much more active and showed a high level of cytotoxic potency, IC50 51 ± 2 and 15 ± 2 nM for 24 h and 48 h of incubation, respectively. The concentrations of digoxin, ouabain and proscillaridin A needed to inhibit [3H]thymidine incorporation into DNA by 50% (IC50) in MDA-MB-231 cells for 24 h of incubation were found to be 124 ± 2 nM, 142 ± 2 nM, and 48 ± 2 nM, respectively. In the present study, we demonstrated that ouabain, digoxin, and proscillaridin A induce apoptosis in MDA-MB-231 cells by increasing free calcium concentration and by activating caspase-3.

Dual effects of ouabain, digoxin and proscillaridin A on the regulation of apoptosis in human fibroblasts.

In this study, we looked at the effect of ouabain, digoxin and proscillaridin A on human fibroblasts. These data show that low concentrations of ouabain, digoxin and proscillaridin A can activate proliferation of human fibroblasts, suggesting that the Na+, K+-adenosine triphosphatase complex may act as a transducing receptor. It was shown that 30 nM ouabain, digoxin and proscillaridin A stimulated an antiapoptotic action by the increase in the level of phosphorylated extracellular signal-regulated kinases (P-ERK 1/2). Ouabain, digoxin and proscillaridin A only at the relatively high concentration of 300 nM increased intracellular Ca2+ concentration, activated caspase-3 and induced apoptosis in human fibroblasts. In terms of reduction in cell viability, antiproliferative and apoptotic activity, these cardiac glycosides rank in the order proscillaridin A >digoxin >ouabain.

Cytotoxic efficacy of a novel dinuclear platinum(II) complex in human breast cancer cells.

Evaluation of the cytotoxicity of a novel dinuclear platinum(II) complex of formula Pt(2)(2-picoline)(4)(berenil)(2) employing a MTT assay and inhibition of [(3)H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that the complex was more of a potent antiproliferative agent than cisplatin. The DNA-binding ability of Pt(2)(2-picoline)(4)(berenil)(2) estimated by an ethidium displacement assay indicated that the complex showed strong specificity for AT base pairs in the minor groove of DNA. Our study showed that Pt(2)(2-picoline)(4)(berenil)(2) was a potent catalytic inhibitor of topoisomerase II in opposition to cisplatin. Pt(2)(2-picoline)(4)(berenil)(2) was found to be a more active inhibitor of collagen biosynthesis than cisplatin. The up regulation of beta(1)-integrin and insulin-like growth factor I (IGF-I) receptor expression by the complex was shown to be accompanied by an increase in the expression of mitogen activated protein kinases in breast cell lines. The phenomenon was related to the increased expression of nuclear factor-kappaB (NuF-kappaBeta) by Pt(2)(2-picoline)(4)(berenil)(2) as shown by the Western immunoblot analysis. Flow cytometric analysis and a fluorescent microscopy assay demonstrated that cell death appeared to result from apoptosis, with the possibility of secondary necrosis. The data presented suggested that Pt(2)(2-picoline)(4)(berenil)(2) impaired growth and metabolism of breast cancer cells more efficiently than cisplatin. These results indicated also the different properties of Pt(2)(2-picoline)(4)(berenil)(2) and cisplatin.

Cytotoxic activity of G3 PAMAM-NH2 dendrimer-chlorambucil conjugate in human breast cancer cells

Evaluation of the cytotoxicity of a novel G3 PAMAM-NH(2) dendrimer-chlorambucil conjugate employing a MTT assay and inhibition of [(3)H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that the conjugate was more potent antiproliferative agent than chlorambucil. It was found that dendrimer-chlorambucil conjugate was more active inhibitor of collagen biosynthesis than chlorambucil. Our experiments carried out with flow cytometry assessment of annexin V binding and fluorescent microscopy assay revealed that PAMAM-CH conjugate inhibited the proliferation of MCF-7 and MDA-231 malignant cells by increasing the number of apoptotic and necrotic cells. The apoptotic effect of PAMAM-CH conjugate was found to be stronger than that caused by chlorambucil.

Small-molecule based delivery systems for alkylating antineoplastic compounds.

Alkylating agents are a major class of anticancer drugs for the treatment of various cancers including hematological malignancies. Targeting alkylating moieties to DNA by attachment of a DNA minor groove binding carrier such as distamycin, netropsin, or Hoechst 33252 reduces the loss of active drug due to reaction with other cell components and makes it possible to direct the alkylation both sequence specifically and regiospecifically. We reported the synthesis and structure–activity studies of amidine analogues of alkylating antineoplastic compounds, which appeared to be a new class of cytotoxic minor groove binders and topoisomerase II inhibitors. Another approach to overcome the toxicity of alkylating agents to normal tissue is to construct a prodrug with lower hydrophobicity and cytotoxicity but is preferentially activated in cancer cells. Overexpression of prolidase in some neoplastic cells suggests that the proline analogue of alkylating agents may serve as a prolidase convertible prodrugs. We have compared several aspects of pharmacological actions of proline analogues of chlorambucil and melphalan in breast cancer cells. The results suggest that prolidase could serve as a target enzyme for the selective action of anticancer agents.

Pyrazolo[4,3-e][1,2,4]triazine sulfonamides as carbonic anhydrase inhibitors with antitumor activity.

A series of sildenafil analogues and aniline substituted pyrazolo[4,3-e][1,2,4]triazine sulfonamides were prepared and evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors and for their anticancer activity against two human breast cancer cell lines (MCF-7, MDA-MB-231). The new compounds were ineffective as CA I inhibitors, poorly inhibited CA II, but were more effective against the tumor-associated isoforms CA IX and XII, with some compounds acting as low nanomolar inhibitors. Evaluation of the cytotoxicity by using an MTT assay, the inhibition of [(3)H]thymidine incorporation into DNA as well as collagen synthesis inhibition, demonstrated that these sulfonamides exhibit cytotoxic effects on breast cancer cell lines ex vivo.

Synthesis, molecular modelling, and antiproliferative and cytotoxic effects of carbocyclic derivatives of distamycin with chlorambucil moiety

New carbocylic analogues of distamycin and netropsin with chlorambucil moieties 5-8 have been synthesised. Data from the ethidium displacement assay showed that these compounds bind in the minor groove of DNA. The observed reduced affinity to AT pairs and increased affinity towards GC sequences of the carbocyclic lexitropsins with chlorambucil moiety 5-8 in comparison with netropsin and distamycin was observed and rationalised by means of molecular modelling techniques. All of the compounds 5-8 showed antiproliferative and cytotoxic effects in the standard cell line of the mammalian tumour MCF-7.

Synthesis and cytotoxic activity of G3 PAMAM-NH2 dendrimer-modified digoxin and proscillaridin A conjugates in breast cancer cells

Abstract The objective of this study was to determine the cytotoxicity, antiproliferative activity, and apoptosis induction activity of two modified glycosides – digoxin and proscillaridin A – conjugated to a generation 3 polyamidoamine dendrimer (G3 PAMAM-NH 2 ) in hu-man breast cancer cells. The results suggest that conjugation with the G3 PAMAM-NH 2 dendrimer enhances the cytotoxicity of modified digoxin and proscillaridin A both in MCF-7 and in MDA-MB-231 breast cancer cells. Additionally, the conjugate-induced apoptosis was significantly greater than apoptosis evoked by free modified digoxin and proscillaridin A.

New pyrazolo[4,3-e][1,2,4]triazine sulfonamides as carbonic anhydrase inhibitors

New sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine were synthesized and investigated as antitumor agents through carbonic anhydrase (CA, EC 4.2.1.1) inhibition. The newly prepared compounds were tested for their anticancer activity against human breast cancer cell lines (MCF-7, MDA-MB-231) using a MTT assay and inhibition of [(3)H]thymidine incorporation into DNA. Preliminary biological studies with several CA isoforms, revealed that the new derivatives were ineffective as CA I and II inhibitors, but they showed activity against tumor-associated enzymes, such as CA IX. The most effective inhibitor against CA IX was compound 8e that exhibited an inhibition constant KI of 13.8nM, and derivatives 8c-8d with moderate activity (8c: KI=25.4nM; 8d: KI=24.5nM). Compounds 8g-8h exhibited acceptable activity (8g: KI=27.7nM; 8d: KI=26.6nM). The detailed synthesis, spectroscopic and biological data are also reported.