Relationship between effects on nucleic acid synthesis in cell cultures and cytotoxicity of 4-demethoxy derivatives of daunorubicin and adriamycin.

Four new derivatives of daunorubicin and two new derivatives of Adriamycin characterized by the absence of the methoxyl groups at the C-4 position have been studied in cell cultures in vitro to establish structure-activity relationships. 4-Demethoxydaunorubicin was 27 to 100 times more active than was daunorubicin when inhibiting the cloning efficiency of exponential-phase HeLa cells and, like daunorubicin, was slightly active on early plateau-phase cells. DNA synthesis in mouse embryo fibroblasts stimulated by fetal calf serum was inhibited equally by the two compounds, although 4-Demethoxydaunorubicin was slightly more active than was daunorubicin when inhibiting RNA synthesis. The beta anomer of 4-demethoxydaunorubicin showed a reduced activity on HeLa cells compared to its alpha anomer, but it was equally active on DNA synthesis. The stereoisomers of 4-demethoxydaunorubicin bearing the inverted configuration in positions 7 and 9 were devoid of significant cytotoxic activity and were only slightly active on DNA synthesis at the doses tested. 4-demethoxyadriamycin and 4-demethoxy-4'-epi-adriamycin were 65 to 500 times more active than was Adriamycin on HeLa cell cloning efficiency and about 10 times more active on DNA synthesis in mouse embryo fibroblasts. Cell uptake in mouse embryo fibroblasts was also investigated for all the new derivatives tested.

Synthesis and antitumor properties of new glycosides of daunomycinone and adriamycinone.

The synthesis of 4'-epi-daunorubicin and of 4'-epi-adriamycin was performed by condensation of 2,3,6-trideoxy-3-trifluoroacetamido-4-O-trifluoroacetyl-alpha-L-arabino-hexopyranosyl chloride with daunomycinone or the protected adriamycinone derivative 17, respectively. Both the alpha and beta anomers were obtained and characterized. All new compounds are biologically active in cultured cells and the alpha anomers display noticeable activity in experimental tumors in mice. Interestingly, 4'-epi-adriamycin (4) appears nontoxic to cultured heart cells up to a concentration of 5 mug/ml.

Changes of activity of daunorubicin, adriamycin and stereoisomers following the introduction or removal of hydroxyl groups in the amino sugar moiety.

The results of a study of the effects of hydroxyl groups at positions, 2, 4 and 6 of the amino sugar on the activity of daunorubicin, adriamycin, and stereoisomers are presented. While the 4'-deoxy derivatives showed a slightly increased biological activity as compared with the parent compounds, the derivatives containing an additional hydroxyl group were less active. It is suggested that the changes in the polarity and in the DNA binding ability of these derivatives are the main factors accounting for the difference in the in vivo activity. The possible relations among the pKa values, the DNA binding properties, and the cellular uptake of the compounds are discussed with particular reference to their therapeutic effectiveness.

Ultrastructural changes produced in cultured, adriamycin-treated myocardial cells.

The ultrastructural evaluation of the early alterations adriamycin-induced on cultured mice heart cells is reported. The major effects are hypertrophy of the sarcoplasmic reticulum and a market increase of the number and total extension of the gap junctions. These findings are discussed in the light of the information available in the literature.

Biological properties of cell lines derived from Moloney virus-induced sarcoma.

Two cell lines derived from a primary MSV-M-induced tumor in a BALB/c mouse were studied. One line (MS-2) was subject only to continuous tissue culture transfer (tct). After 21 tct, MS-2 cells produced progressive tumors (MS-2 tumors) in syngeneic hosts. The second cell line (MS-2T) was established by cultivation of a MS-2 tumor. The ability to produce progressive tumors decreased with increased number of tct, in both cell lines. The virus content of MS-2 and MS-2T cells was very low, as shown by uridine incorporation and electron microscopy. Immmunofluorescence tests demonstrated that antigens different from the viral MSV-M antigens were present on the cell lines, and that antigenic changes occurred with increased number of tct. Serum of mice bearing progressive MS-2 tumors reacted with MS-2T cells when these cells produced progressive tumors and did not react with MS-2 cells when they produced regressing tumors. MS-2 cells producing regressing tumors reacted with serum from mice in which the MS-2 tumor had regressed and with serum from mice immunized with MS-2T cells at late tct when they were poorly oncogenic. The antigenic changes seemed, therefore, to parallel the decrease of malignancy. A chromosomal analysis carried out on MS-2 and MS-2T cells, when both produced progressive tumors, showed a modal number of 48 and 44, respectively. MS-2T cells showed a large acrocentric chromosome. In contrast, the MS-2 cells at late tct, when they gave regressing tumors, showed a modal number of 60 and a wide range of distribution of chromosome number.

Actin behaviour in doxorubicin treatment.

The inhibition of neural tube closure in early chick embryos explanted in vitro by New's technique was used as a model to study the possible involvement of actin polymerization during doxorubicin treatment. The investigation was performed with indirect immunofluorescence staining, fluorescence staining with NBD-Ph, and DNase I inhibition activity. 8 x 10(-6) M doxorubicin induces alteration in the distribution of actin fluorescence on neural tube and in CEF cell cytoskeleton, and an appreciable increase in the critical concentration of monomeric actin as measured in cell extracts.

Dose-dependence of doxorubicin effect on actin assembly in vitro.

Doxorubicin (Adriamycin), one of the most potent antibiotics used in tumor chemotherapy, shows many undesirable side effects. We studied the effect of different drug concentrations on the biochemistry of cell motility and, in particular, on potassium-induced actin polymerization. It is well known, in fact, that the actin aggregational status could dramatically influence many cell motility manifestations. Our results clearly show that stoichiometric and substoichiometric amounts of doxorubicin negatively influence actin polymerization by inhibiting both the filament growth and the polymer amount at steady-state; the balance between the two different effects seems to be in relation to the drug concentration. The obtained results could explain some of the doxorubicin effects previously observed in vivo.

The effects of doxorubicin on embryonic spinal motoneurons cultured in vitro: cytoskeletal response.

The interaction of doxorubicin with tubulin in spinal motoneuronal cells cultured in vitro has been examined by laser scan microscopy with an immunocytological double staining technique, and by biochemical analysis. Results obtained by laser scan microscopy indicate that doxorubicin (2 x 10(-8) M) was able to induce fragmentation or disappearance of microtubules in motoneuronal cells. Motoneurons were identified by their positivity to anti-CAT antibody. This observation was then studied by biochemical analysis on purified calf brain tubulin. Even treatment with a high dose of doxorubicin (1.2 x 10(-5) M) does not impair tubulin polymerization. The finding supports the possibility that doxorubicin interacts with MAPs or other polypeptides present in the cytomatrix that coassemble with tubulin and are necessary for microtubule formation.

Doxorubicin affects actin assembly in vitro.

In vitro experiments on actin polymerization in the presence of doxorubicin show that the rate of salt-induced actin assembly is negatively affected by the drug. The decreased amount of actin monomers keeping their ability to self-interact to give F actin (microfilaments) probably explains the reduction of assembly value. Drug action is dose-dependent and various discrepancies are explained by the limitations of the techniques used.