[Anticancer drug research in Hungary, 1950-2000]. / Daganat-kemoterápiás kutatások Magyarországon az 1950−2000 közötti években.

The present review about the history of anticancer drug research in Hungary intends to call attention to the importance of studies on their mode of action. Several lines of evidence suggest that clinically usable oncopharmacological properties could be revealed by this way. Among the numerous compounds certain alkylating sugar alcohols and 2'-deoxyuridine derivatives were submitted to detailed investigations concerning their mode of action. Myelobromol with selective action on the myeloid elements of bone marrow has been justified for its application in chronic myeloid leukemia therapy and also in bone marrow ablation before transplantation. Mitolactol is able to cross bloodbrain barrier, consequently could control certain brain tumors. 5-etil-2'-deoxyuridine by reducing dihydropyrimidine dehydrogenase activity is able to increase 5-fluorouracil concentration in the blood, resulting in improved antitumor effect. In contrast, 5-hexil-2'-deoxyuridine, as an inhibitor of glycoconjugate pathway by reducing heparan sulfate production, has the ability to prevent metastasis. Noteworthy, the remarkable effects of vinca alkaloids, antiestrogens, and GNRH analogues were also presented in this review.

E-cadherin-dependent intercellular adhesion enhances chemoresistance.

E-cadherin, an intercellular adhesion molecule, is important in cell growth and differentiation. Adhesion between cells is thought to decrease as cancers develop and disseminate. Knowledge of the effect of cell adhesion on proliferation and chemosensitivity may help individualize cancer treatment. Lovo and MCF-7 cells, which express E-cadherin, and PC-3 cells, which do not, were used in this study. Proliferation and chemosensitivity were measured in two-dimensional (2-D) culture and three-dimensional (3-D) culture. Protein and mRNA expression of E-cadherin, catenin, and cyclin-dependent kinase inhibitors were determined. Growth of Lovo and MCF-7 but not PC-3 cells was markedly suppressed in 3-D relative to 2-D. MCF-7 cells express high levels for E-cadherin, catenin, and p27 in 3-D, but catenin and p27 expression was decreased by exposure to anti-E-cadherin neutralizing antibody. Chemosensitivity of PC-3 was similar in 2-D and 3-D, but chemosensitivity of Lovo and MCF-7 was less in 3-D than 2-D. Moreover, the presence of anti-E-cadherin antibody increased chemosensitivity of MCF-7 in 3-D. E-cadherin affected the regulation of cell proliferation and differentiation, and decreased chemosensitivity. Chemosensitivity of cancer is affected by the state of cell adhesion and expression of intercellular adhesion molecules. Consideration of intercellular adherence characteristics in different chemosensitivity tests is likely to improve their reliability.

Reversal of multidrug resistance by novel nitrophenyl pyrones, SNF4435C and D.

SNF4435C and D, novel immunosuppressants produced by a strain of Streptomyces spectabilis, were examined for their reversing effects in vitro on various multidrug-resistant (MDR) tumor cells overexpressing P-glycoprotein. These two compounds in the range of 3-10 microM completely reversed the resistance of MDR variant cells, mouse leukemia P388 cells [vincristine (VCR)-resistant P388/VCR and adriamycin (ADM)-resistant P388/ADM], human myelogenous leukemia K562 cells (VCR-resistant K562/VCR and ADM-resistant K562/ADM) and human ovarian cancer A2780 cells (ADM-resistant AD(10)), against VCR. Both compounds moderately potentiated the sensitivity of the MDR cells to ADM but the reversal was not complete. SNF4435C and D significantly increased the intracellular accumulation of VCR in AD(10) cells as potently as verapamil, cyclosporin A (CysA) and FK506, whereas the compounds exerted no effect on the accumulation of VCR in the drug-sensitive parent cells. Moreover, SNF4435C improved the chemotherapeutic efficacy of VCR in the treatment of P388/VCR-bearing mice. When 10 mg/kg SNF4435C was administered intraperitoneally to the mice concurrently with 0.2 mg/kg VCR for every 5 days, a treated/control (T/C) value of 143% was obtained. These results suggest that the compounds are useful candidates or tools for MDR modification in cancer chemotherapy.

Apoptotic and mitotic activity in squamous cell carcinoma cells after combined modality treatment with gamma-irradiation and dibromodulcitol.

A-431 squamous cell carcinoma cells were treated in vitro with either 4 Gy radiation of 15 (or 45) microg/ml dibromodulcitol (DBD), as well as with combined 4 Gy irradiation and DBD, with the latter as either a pretreatment or post-treatment. DBD alone or in combination with radiation had a greater effect on cell proliferation than the effect of radiation alone. The difference is due to a higher level of apoptosis induced by DBD, especially in conjunction with radiation. Such a combination may therefore be useful in the treatment of squamous cell carcinoma, which in general responds poorly to radiation therapy.

The biological activity of cisplatin and dibromodulcitol in combination therapy.

The efficacy and modes of action of dibromodulcitol (DBD) and cisplatin (CDDP) were studied in several model systems. Combination treatments produced a longer survival time in mice bearing P388 solid lymphomas than either of the drugs alone. In the human metastatic melanoma HT-168 xenograft model the combined application of DBD and CDDP was also very effective, inducing a reduction in the number and volume of metastatic nodules. For V79 spheroids, DBD was mainly cytotoxic against the internal, quiescent cells, whereas cisplatin primarily killed cells in the proliferating, external regions of the spheroids. When combined, the drugs appeared to act synergistically throughout the spheroids. Studies on plasmid DNA showed that CDDP primarily generates cross-links, whereas single-strand breaks were dominant after DBD treatment. Upon using an assay for cleavage by restriction nuclease, antagonistic action of DBD and CDDP in combination may occur, nevertheless more strand breaks were always observed in these samples. These results suggest that the efficacy of combined DBD and CDDP is in part a result of 'spatial cooperation' by the drugs (i.e. affecting different cells) and in part the result of DNA damage produced by the combination treatments.

Functionally active homodimer of P-glycoprotein in multidrug-resistant tumor cells.

P-glycoprotein plays a key role in multidrug resistance of tumor cells. In order to elucidate the possible quarternary structure/function relationship of P-glycoprotein, we treated multidrug-resistant human leukemia K562/ADM cells with the crosslinking reagent, disuccinimidyl suberate. In addition to 180K P-glycoprotein, a 340K protein was immunoprecipitated with an anti-P-glycoprotein monoclonal antibody, MRK-16. The 340K protein is most probably a dimeric P-glycoprotein, since only the 180K P-glycoprotein was immunoprecipitated with MRK-16 when K562/ADM cells were treated with the cleavable crosslinking reagent, dithiobis(succinimidylpropionate), and analysed under reduced conditions. The dimeric P-glycoprotein was photolabeled with [3H]azidopine like the 180K monomeric P-glycoprotein and the photolabeling was inhibited by excess amount of vincristine and verapamil. The dimeric P-glycoprotein could be a functionally active form of the protein involved in the transport of antitumor agents.

Antitumor activity of siwenmycin: a novel anthracycline antibiotic.

A novel anthracycline antibiotic, siwenmycin, isolated from the culture of Streptomyces galilaeus var. siwenesis, was examined for its antitumor activities against P388, K562, B16-F10, HeLa, HEp-2 and Lewis lung carcinoma cell lines. The results showed that siwenmycin was effective against P388, K562, HeLa and HEp-2 tumor cell lines in vitro, and significantly inhibited the growth of the Lewis lung carcinoma cell line in vivo. Siwenmycin could also suppress spontaneous and artificial pulmonary metastases of B16-F10 and Lewis lung carcinoma cell lines in C57BL/6 mice. The inhibitory effect of siwenmycin on spontaneous pulmonary metastasis of Lewis lung carcinoma in C57BL/6 mice was even stronger than that of adriamycin (ADM), which is, at present, commonly used in clinical practice. Furthermore, the double-labeling test used in this study has verified that siwenmycin can inhibit cellular RNA synthesis at about one tenth the concentration required to inhibit DNA synthesis to the same degree, indicating that the antitumor mechanism of siwenmycin also differs from that of ADM. The acute toxicity of siwenmycin was very low, and it was as effective in vivo as in vitro, suggesting that this newly found antibiotic should be studied for possible clinical antitumor applications.

The diverse modification of N-butyl-N-(4-hydroxybutyl) nitrosamine induced carcinogenesis in urinary bladder by dibromodulcitol and dianhydrodulcitol.

The effects of intravesical therapy with adriamycin, 1.6-dibromo-1.6-dideoxydulcitol (DBD) or with 1.6-dianhydrodulcitol (DAD) on bladder carcinogenesis were investigated in rats. To induce premalignant lesions in the urinary bladder female Sprague-Dawley rats received 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in their drinking water for 4 weeks and then post-treated intravesically with one of the antitumor drugs and examined once a week for 3 months. These antitumor drugs in healthy rats (i.e. without the pre-administration of BBN) did not cause any significant morphological changes in the urinary bladder after intravesical application once a week for 3 months. In the applied dose BBN alone induced only premalignant lesions in the urinary bladder. However, neoplastic lesions occurred in the groups treated with BBN and adriamycin (9 papillomas and 3 carcinomas in 14 animals). Similarly intravesical application of DBD after BBN administration resulted 5 carcinomas among the 11 animals. On the contrary no urinary bladder tumor was found in the animals treated with BBN and DAD. As DAD is one of the conversion products of DBD it is conceivable that the difference between DBD and DAD action may be due to the formation of other solvolytic product from DBD than DAD.

Potentiation of BCNU-induced cytotoxicity and sister chromatid exchanges by dibromodulcitol in vitro.

Dibromodulcitol (DBD) is an anticancer agent that is cytotoxic against animal and human brain tumors in vivo. Clinical trials of combination therapy with radiation, DBD, and a nitrosourea have shown some efficacy, but the mechanisms that lead to enhanced cytotoxicity are poorly understood. We investigated the effects of pretreatment with DBD on cell survival and sister chromatid exchange (SCE) caused by subsequent treatment with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) in 9L rat brain tumor cells. Pretreatment of 9L cells for 24 hr with 5 microM DBD potentiated the cell kill produced by a 1-hr treatment with BCNU; the dose enhancement ratio was 1.7 at the 10% survival level. Treatment of 9L cells for 24 hr with 1 microM DBD induced 39 +/- 12 SCEs/metaphase. There was a 50-75% increase in BCNU-induced SCEs, compared with BCNU alone, after a 24 hr pretreatment with DBD. Thus DBD potentiation of BCNU cytotoxicity appears to be related to increased DNA damage.

In vitro chemosensitivity of brain tumors to cisplatin and its analogues, iproplatin and carboplatin.

The human tumor stem-cell assay was used to investigate the in vitro chemosensitivity of 27 evaluable samples to cisplatin and its analogues, iproplatin and carboplatin, as well as to BCNU, teniposide, vindesine, and dibromodulcitol. All agents exhibited some antitumor activity with the exception of dibromodulcitol (zero response out of 19 evaluable samples). Vindesine, BCNU, and carboplatin were the three most active compounds, with response rates of 29%, 23%, and 22%, respectively. There was a lack of complete cross-resistance between carboplatin and cisplatin as well as between carboplatin and BCNU. Our data suggest that clinical studies with carboplatin and combinations of vindesine plus cisplatin and its analogues may be worthwhile.

Potentiation of antimetabolite action by dibromodulcitol in cell culture.

The postulation that the activity of key enzymes that reveal marked increases should be potential targets for anticancer chemotherapy (47) was supported by new evidence on the alterations of CDP reductase, CTP synthetase and OMP decarboxylase in hepatoma 3924A cell cultures. Inhibitors of these enzymes (VF-122, acivicin, pyrazofurin) and that of IMP dehydrogenase (tiazofurin) efficiently killed hepatoma 3924A cells in culture, as demonstrated by the clonogenic assay. Acivicin, pyrazofurin, tiazofurin and VF-122 were lethal against tumor cells in the exponential phase of growth with IC50 of 1.5, 5, 10 and 4.5 microM, respectively. All these antimetabolites exhibited cytotoxicity preponderantly against exponential-phase cultures, indicating that all the four drugs belong to Class II (phase-specific agents) in the Kinetic Classification of Anticancer Agents (38). Dibromodulcitol, a bifunctional alkylating agent, revealed cycle-specific cytotoxicity (Class III agent) against hepatoma 3924A, yielding IC50 values of 2.3 and 5.5 microM for exponentially and stationary growing cells, respectively. Using isobologram analysis on the survival data of 3924A cells, synergistic interaction was observed when DBD in combination with acivicin, pyrazofurin and tiazofurin was examined. DBD in combination with VF-122 exhibited additive lethality against hepatoma cells in culture. The synergistic and additive cytotoxicity in combinations of DBD with these antimetabolites was accompanied by the concurrent depletion of ribonucleotide and/or deoxyribonucleotide pools. The synergistic biological results of drug combinations of acivicin with DBD can be accounted for by the action of acivicin in inhibiting CTP synthetase, resulting in a synergistic decrease in CTP content, and by inhibition of DNA synthesis caused by DBD. The synergistic and additive depletion of UTP, CTP, dTTP and dCTP pools in the combinations of DBD with pyrazofurin may be responsible for the synergistic lethality of these combinations. Synergism, in terms of pool depletion, was observed for GTP and dCTP; summation was detected for dGTP when DBD and tiazofurin were given concurrently. The synergistic cytotoxicity of this drug combination may be a consequence of these alterations. The additive lethality of DBD-VF-122 drug combinations was reflected in the additive elevations of the ribonucleoside diphosphate concentrations. These observations indicate that treatments based on the Kinetic Classification and on the biochemical targeting of the drug should have an impact on the design of in vivo chemotherapy.

Cytochemical characterization of Yoshida sarcoma cells resistant to dibromodulcitol.

Two Yoshida ascites sarcoma cell populations, one of them originally sensitive and another rendered resistant to the alkylating agent dibromodulcitol (DBD), were compared for doubling time, labeling index, survival time, morphological features, cellular DNA content and modal DNA value. Cross-resistance studies were carried out on bilaterally growing solid tumours derived from the sensitive and resistant Yoshida cell populations. Glycogen-containing granules appeared in the cytoplasm of the DBD-resistant sarcoma cells; these were not present in the sensitive tumour. The growth parameters of the sensitive tumour were throughout the 10-day observation period characteristic of rapidly proliferating tumours, whereas those of the DBD-resistant tumour was characteristic of slowly proliferating systems. As compared to the mean DNA values for normal somatic cells (lymphocytes), those found for both tumours indicated that the cells were hyperdiploid aneuploid. The DNA stem line in the resistant tumour proved to be significantly smaller than that in the sensitive tumour. These results indicate that a reduction of malignancy in the Yoshida tumour is associated with a developed resistance to dibromodulcitol. The resistant tumour showing cross-resistance to dianhydrogalactitol, diacetyl-dianhydrogalactitol and to nitrogen mustard retained full sensitivity to cyclophosphamide and adriablastin (Adriamycin), and proved to be collaterally sensitive to vincristin.

Identification of guanine and adenine adducts in DNA alkylated by dibromodulcitol in vitro and in vivo.

DNA reacted with dibromodulcitol in neutral solution yielded 3- and 7-alkyl substituted purines after hydrolysis at neutral pH-value at 37 degrees C. The alkylated products were identified by mass spectrometry and by comparison of their UV absorption spectra and chromatographic properties on thin-layer chromatography (TLC) and various columns with those of the corresponding galactitylpurine derivatives obtained by synthetic route from alkylation of the appropriate nucleic bases or nucleosides. The labelled alkylpurines occurring in DNA of Yoshida tumour cells treated with [3H]dibromodulcitol in vivo were also identified by co-chromatography of labelled DNA hydrolysate with synthetic 3- and 7-alkyl substituted purines. On the basis of the same chromatographic behaviour 3-(1-deoxy-3,6-anhydrogalactit-1-yl)adenine, 7-(1-deoxygalactit-1-yl)guanine, 7-(1-deoxy-3,6-anhydrogalactit-1-yl)guanine and 1,6-di(guanin-7-yl)-1,6-dideoxygalactitol were identified as main alkylated products in tumor cell DNA after in vivo treatment with dibromodulcitol.

Effect of phenobarbital on the toxicity and antitumor activity of dibromodulcitol and dianhydrogalactitol.

5-Ethyl-5-phenylhexahydropyrimidin-2,4,6-trione (phenobarbital) pretreatment significantly decreased the acute toxicity of 1,6-dibromo-1,6-dideoxygalactitol (dibromodulcitol) in H/Riop-Swiss mice and Wistar rats. Toxicity of dianhydrogalactitol, however, was not influenced to a considerable measure. Phenobarbital did not alter in itself the growth rate of the tumours examined. It significantly reduced, however, the antitumor activity of dibromodulcitol in NK/Ly-mouse lymphoma and Yoshida rat tumor. In contrast, the cytostatic effect of dianhydrogalactitol was not influenced at all by phenobarbital. The dissimilar effects of phenobarbital on the toxicity and anti-tumour activity of dibromodulcitol and dianhydrogalactitol are probably due to differences both in their metabolism and pharmacodynamics.

The effect of dibromo-dulcitol and dianhydro-dulcitol (galactitol) on RNA synthesis in ascites tumor cells.

The mechanism of action on RNA synthesis of anticancer dibromo-dulcitol (DBD, NSC-104800) and dianhydro-dulcitol (DAD, or elsewhere dianhydrogalactitol, DAG, NSC-132313) was investigated. Rats, bearing Yoshida or Novikoff hepatoma ascites tumor cells sensitive to these drugs were treated with doses equivalent to half the LD50 value. Nucleolar RNA (noRNA) and nuclear RNA (nRNA) were pulse labelled with 3H-uridine, isolated and fractionated on sucrose density gradient. After 18 h treatment with either drug and after 3 h with DAD noRNA synthesis increased and the rate of ribosomal RNA (rRNA) precursor processing was enhanced. Investigation of low-molecular weight nRNAs (LMW nRNAs) (separated by polyacrylamide gel electrophoresis) showed increased synthesis and/or accumulation of RNA species (5S RNA, uridylic acid rich RNAs) related to rRNA synthesis. The tritium labelled drugs were bound to distinct fractions of nRNA, separated by sucrose density gradient ultracentrifugation, both in vivo and in vitro. This fact may be explained by the formation of intra-, or intermolecular crosslinking of pre-messenger RNA. The enhanced RNA synthesis might be interpreted as an alteration in the functions of nuclear proteins, involved in the regulation of gene transcription and processing of RNA precursors.

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.

Chromatin proteins as a possible target for antitumour agents: alterations of chromatin proteins in dibromodulcitol treated Yoshida tumors.

The effect of dibromodulcitol (DBD) on Yoshida sarcoma chromatin components has been investigated. Measurements on the radioactivity of nuclear components after in vivo treatment with [3H]DBD for 1 h indicated preferential drug binding to the high molecular weight component of the nuclear residual acidic protein (non-histones) and also to Histone 1 (H1) (very lysine rich, F1). Two-hour DBD treatment resulted in partial degradation and reduced [3H]leucine incorporation into the same fractions of chromatin. However, 6 h after DBD treatment, the synthesis of the degraded chromatin proteins began and by 24 h was completed. During the same treatment period the composition of chromatin showed a remarkable alteration; 2 h after DBD treatment the amount of the nuclear residual acidic proteins relative to DNA decreased by approx. 50%, but returned to control value 24 h after drug treatment. This in conjunction with the data on [3H]leucine incorporation suggests that certain chromatin proteins are degraded and subsequently newly synthesised after DND treatment resulting in an exchange of chromatin components. The formation of a nucleohistone complex between H1 and DNA was inhibited by pretreatment of H1 and DBD, dianhydrodulcitol (DAD) and bischloroethylnitrosourea (BCNU).

Chromosomal analysis on Yoshida tumor cells sensitive and resistant to dibromodulcitol.

Yoshida ascites tumor in Wistar rats was rendered resistant to dibromodulcitol. The modal number of chromosomes in the parent line was reduced from 39 to 36 in the resistant one. The parent tumor was a mixed cell population, containing large subtelocentric (LST) chromosome in 65 per cent of the cells, while in the remaining 35 per cent the LST chromosome was absent. In the resistant tumor line, however, the LST marker chromosome was present only in a few cells. The resistant line preserved this new karyotype throughout the 5 months of passaging even without dibromodulcitol, however, parallel to the loss of resistance this tumor line regained the original number of chromosomes, i. e. 39. Selection of a cell population with reduced number of chromosomes seems to be associated to the development of drug resistance.