Antitumor activity and induction of apoptosis by water-soluble derivatives of 7 beta-hydroxycholesterol in human colon carcinoma cell lines.

At present, cancer therapy of solid tumors, such as lung and colorectal cancer, is unsatisfactory. Recently, oxygenated sterols have shown selective cytotoxicity against tumor cells. In this study, the cytotoxicity of 7 beta-hydroxycholesterol (7 beta HC) and two water-soluble derivatives of 7 beta HC, i.e. 7 beta HC-bis-hemisuccinate [disodium salt] (7 beta HC-HS) and 7 beta HC-bis-hemisuccinate-diethanolaminoate (7 beta HC-EA), was determined in DLD-1, KM20L2, HCT-116, HT-29 and SW620 colon carcinoma cell lines using a cell count assay. IC50 values of the two water-soluble derivatives were, on the whole, comparable to 7 beta HC lying in the range of 3-10 microM. In addition, the water-soluble derivatives were able to induce apoptosis in the examined DLD-1 and KM20L2 colon carcinoma cell lines in contrast to the parent compound 7 beta HC, as shown by DNA fragmentation, by the cleavage of DNA repair enzyme poly(ADP) ribose polymerase (PARP), and by the proteolytic cleavage of caspase-3 (CPP32). Due to the improved water-solubility of 7 beta HC-HS and 7 beta HC-EA and their promising antitumor activity in vitro, animal studies in suitable tumor models are warranted.

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.

rhG-CSF affects genes involved in mitogen signalling and early gene expression in the ovarian cancer cell line HEY.

The ovarian adenocarcinoma cell line HEY was used as an in vitro model to study the influence of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on epithelial tumours such as ovarian cancer. Serum-starved cells were treated with rhG-CSF in a time- and dose-dependent manner. Cell proliferation, measured as cell division and DNA synthesis, was stimulated about 40% by rhG-CSF. After harvesting, cells were examined for the presence of G-CSF receptor (FACS analysis and RT-PCR), as well as for expression of genes involved in mitogen signalling (ERKs, JNKs) and early gene expression (c-jun). rhG-CSF affected mitogen-activated pathways and was receptor-mediated if the G-CSF receptor was present. After rhG-CSF induction, Janus N-terminal kinases (JNK 1 and 2) were simultaneously increased in the cytosol, up to 30-fold as measured by Western blotting), whereas ERK 1 and 2 accumulated maximally by 2.5-fold 1 hr after rhG-CSF induction. c-Jun was up-regulated strongly by this cytokine at the translational level. Our data suggest that rhG-CSF affects genes involved in mitogen signalling and early gene expression in solid tumours. We also noted the presence of G-CSF receptor on ovarian cancer cell lines.

Preparation, characterization and in vitro efficacy of albumin conjugates of doxorubicin.

One strategy for improving the antitumor selectivity and toxicity profile of antitumor agents is to design drug carrier systems with suitable transport proteins. Thus, four maleimide derivatives of doxorubicin were bound to thiolated human serum albumin which differed in the stability of the chemical link between drug and spacer. In 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 albumin 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 with that of the free drug (using the BrdU-(5-bromo-2'-deoxyuridine)-incorporation assay and tritiated thymidine incorporation assay respectively, IC50 approximately 0.1-1 microM) whereas conjugates with the amide derivatives showed no or only marginal activity. These results demonstrate that antiproliferative activity depends on the nature of the chemical bond between doxorubicin and carrier protein. Acid-sensitive albumin conjugates are suitable candidates for further in vitro and in vivo assessment.