Value of 31P NMR spectroscopy in predicting the response of a xenografted human hypopharynx carcinoma to irradiation.

PURPOSE: An early indicator of tumor sensitivity to irradiation could provide useful information on the effectiveness of therapy and may facilitate more individual designs of treatment protocols. The aim of the present study was to evaluate the potential of in vivo 31P nuclear magnetic resonance spectroscopy in predicting the response of a xenografted human hypopharynx carcinoma to radiotherapy. METHODS: The tumor had been serially heterotransplanted to athymic mice. 31P NMR spectra were collected before and at four intervals (24, 48, 72, and 120 h) after irradiation with 15 Gy or 30 Gy. Alterations of phosphorus metabolism were compared with the growth delays, the histological appearance, and the mitotic activity of the treated tumors. RESULTS: Radiation with 30 Gy induced increases of the phosphodiester level (P < 0.001) as well as of the tumor pH (P < 0.05) and decreases of the phosphomonoester level (P < 0.001) within 48 h. The changes clearly preceded measurable tumor responses and were accompanied by severe histological destruction and marked depression of mitotic indices. However, none of these spectral alterations was significantly correlated with individual delays of tumor growth. The only parameters allowing a prediction of radiation-induced tumor responses were the pre-treatment levels of phosphomonoesters and -diesters. The 31P NMR spectroscopic changes observed after therapy with 15 Gy were either unsystematic or insignificant. CONCLUSIONS: Pretreatment levels of tumor phospholipids were indicative of radiosensitivity in the xenografted human hypopharynx carcinoma investigated here. However, since phosphorus metabolism varies considerably among different tumor lines, it seems unlikely that there exists a uniform 31P NMR spectroscopic parameter predicting tumor response to radiation therapy.

Antitumor activity of titanocene dichloride in xenografted human renal-cell carcinoma.

Titanocene dichloride [(C5H5)2TiCl2] is a new-developed organometallic antitumor agent which is currently being investigated in clinical trials of phases I and II. In the present study, it was tested for antitumor activity in human renal tumors either growing as monolayers in vitro or as xenografts in athymic mice. For comparison, approved cytostatic drugs (in vitro, vinblastine and 5-fluoro-2'-deoxyuridine; in vivo, cyclophosphamide, vinblastine, and 5-fluorouracil) were administered in vitro and in vivo at equivalent or equitoxic dose levels, respectively. Under in vitro conditions, titanocene dichloride was active only moderately. When it was applied at peak plasma level of 10(4) mol/l, it induced cell growth inhibitions by 25-50% in all KTCTL cell lines investigated (KTCTL-1M, KTCTL-2, KTCTL-26A, KTCTL-30, KTCTL-84). In the N-U 2 carcinoma cell strain it was more effective and caused cell growth inhibitions of 70-80% at the 10(-4) mol/l level, the IC50 value amounting to 5 x 10(-6) mol/l. When titanocene dichloride was applied intraperitoneally (i.p.) according to the Q3Dx5 and Q2Dx5 regimens and investigated in the human renal-cell carcinoma N-U 2 growing as xenograft in athymic mice, it brought about significant and dose-dependent growth reductions by 50-75% in relation to untreated controls, whereas cyclophosphamide given as single bolus injection and vinblastine administered both as single and triple doses were slightly less effective in this xenograft. MKT 4 and MKT 5, two formulations of titanocene dichloride which are currently used in clinical trials, showed similar efficacy as titanocene dichloride towards the N-U 2 renal-cell carcinoma xenograft. In the heterotransplanted N-U 26 carcinoma, titanocene dichloride induced relative growth reductions by 50-56% and was similarly active as cyclophosphamide, but less effective than vinblastine applied as a single dose. Titanocene dichloride was again significantly active in the KTCTL-1M carcinoma xenograft and caused relative growth reductions by 50-65%. In the case of the MRI-H 121 renal sarcoma xenograft, however, the organometallic compound showed an only marginal activity which was surpassed by cyclophosphamide, vinblastine and 5-fluorouracil, all three drugs inducing significant relative growth inhibitions by 50-88%. These results confirm a significant and remarkable antitumor activity of titanocene dichloride in three out of four human renal tumors xenografted to athymic mice and suggest that clinical studies of phase II with titanocene dichloride towards renal-cell carcinoma in human patients should be done in the near future.

The alkylator treosulfan shows activity towards human renal-cell carcinoma in vivo and in vitro.

Treosulfan (L-threitol-1,4-bismethanesulfonate, Ovastat) was tested on human renal tumor cells growing as xenografts in athymic nude mice and as monolayers in vitro, in comparison with clinically used cytostatic drugs (in vivo, cyclophosphamide, vinblastine, and 5-fluorouracil; in vitro, vinblastine and 5-fluoro-2'-deoxyuridine) which were administered at equitoxic or equivalent dose levels, respectively. Four human renal tumor xenografts (N-U 2, N-U 26, MRI-H 121, KTCTL-1M) were investigated in vivo, and seven renal tumor cell lines (KTCTL-1M, KTCTL-2, KTCTL-26A, KTCTL-30, KTCTL-84, MRI-H 121, N-U 2) under in vitro conditions. The investigations of the four human renal tumor xenografts revealed that treosulfan is capable of inducing pronounced growth inhibitions ranging from 60-100% in comparison with untreated control tumors. In the xenografted renal-cell carcinoma KTCTL-1M, treosulfan administered at the highest dose level (1 x 3,500 mg/kg) even effected a complete remission lasting for more than three weeks in all animals treated with this dose. It was more effective in the N-U 2 carcinoma growing in vivo than the comparative compounds cyclophosphamide and vinblastine. In the heterotransplanted renal-cell carcinoma N-U 26, treosulfan showed a similar activity as the two established cytostatic drugs tested whereas, in the renal sarcoma MRI-H 121, both cyclophosphamide and vinblastine were slightly more effective than treosulfan. In four renal-cell carcinomas growing as monolayers in vitro (KTCTL-1M, KTCTL-2, KTCTL-84, N-U 2), treosulfan induced cell growth inhibitions by about 50% at peak plasma concentration in comparison with untreated control cultures. The IC50 values ranged from 5 x 10(-6) to 10(-4) mol/l in all seven monolayer cultures investigated 5-Fluoro-2'-deoxyuridine (floxuridine) was similarly active in vitro as treosulfan with respect to the molar concentrations inducing growth inhibition and to the IC50 values, whereas vinblastine was more effective than treosulfan in most of the human renal tumor cell monolayers investigated. These results reveal the remarkable antitumor efficacy of treosulfan towards human renal-cell carcinomas, especially under in vivo conditions. This activity was similarly high or even better than in cyclophosphamide and vinblastine. The in vitro data obtained in monolayer cultures also confirmed the remarkable antiproliferative activity of treosulfan in renal tumor cells, but did not mirror very well the pattern of antitumor activity observed in vivo.

Antitumor activity of titanocene amino acid complexes.

Seven ionic titanocene alpha-amino acid (aa) complexes [(C5H5)2Ti(aa)2]2+[X]2- with aa = glycine, L-alanine, 2-methylalanine, D-L-phenylalanine, D,L-4-fluorophenylalanine and X = Cl or AsF6, were investigated for antitumor activity against fluid Ehrlich ascites tumor growing in CF1 mice. These complexes are the first stable model compounds of titanocene units with protein components, synthesized from a water-like, methanolic medium. All titanocene amino acid complexes induced antitumor activity which was manifested by maximum cure rates ranging from 30 to 70% and increases in life span from 78 to 276% in comparison with untreated control animals. The complexes containing chloride as anion X were more effective than the hexafluoroarsenate derivatives, which surprisingly showed a low substance toxicity. In all cases, the antitumor activity of the ionic titanocene amino acid complexes tested was less pronounced than that of the neutral parent compound [(C5H5)2TiCl2].

Antitumor activity of treosulfan in human lung carcinomas.

Treosulfan (L-threitol 1,4-bismethanesulfonate, Ovastat) is an alkylating agent and a structural analogue of busulfan. It has been established in the clinical chemotherapy of human ovarian carcinomas for several years and has additionally been shown to be effective against xenografted human breast carcinomas. No other human carcinoma is yet known to be sensitive to treosulfan. The present study confirms the pronounced and significant antitumor activity of treosulfan against heterotransplanted human lung carcinomas of both the small-cell and the non-small-cell type. Treosulfan reduced the growth of all four small-cell lung carcinomas that were investigated in a significant manner. It was even more active than equitoxic doses of the clinically approved cytostatics ifosfamide, cisplatin, and etoposide toward three of them and induced long-lasting growth reductions (60-98% of control tumor size) corresponding to partial and nearly complete remissions. In the case of the nine non-small-cell lung carcinomas investigated, treosulfan effected significant growth inhibition of more than 50%, again in all of them, and was more active than the comparative compounds ifosfamide, mitomycin C, and cisplatin at least in one of four epidermoid lung carcinomas, one large-cell carcinoma, and one of three lung adenocarcinomas. These results are remarkable and unexpected, and the present study should be followed rapidly by phase II clinical trials of treosulfan against human lung carcinomas of both the small-cell and the non-small-cell type.

5-Fluorouracil metabolism and cytotoxicity after pre-treatment with methotrexate or thymidine in human hypopharynx and colon carcinoma xenografts: a 19F-nuclear magnetic resonance spectroscopy study in vivo.

The metabolism of 5-fluorouracil (5-FU) was monitored non-invasively in two xenografts, a hypopharynx carcinoma and a colon carcinoma (CSM) by 19F-magnetic resonance spectroscopy following an i.v. bolus injection of 130 mg kg-1 5-FU. Both the level of fluoronucleotides (FNuc) and the tumor growth delay were significantly higher in the CSM colon carcinoma than in the hypopharynx carcinoma (both parameters, P < 0.001). Administration of 100 mg kg-1 methotrexate (MTX) at 15 h before treatment with 5-FU caused a significantly increased conversion of 5-FU to FNuc in both tumors (P < 0.05) as compared with the application of 5-FU alone. However, only in the CSM tumor was a significantly increased growth delay (P < 0.01) observed. Pre-treatment of both xenografts with 400 mg kg-1 thymidine enhanced the conversion of 5-FU to FNuc in both tumors. In the CSM tumour this treatment modality caused a significantly (P < 0.05) higher growth delay as compared with the results obtained with 5-FU alone, whereas in the hypopharynx carcinoma the additional application of thymidine caused no significant change in tumor growth. It is known that both thymidine and MTX can reduce the DNA-directed cytotoxicity of 5-FU, whereas the RNA-directed cytotoxicity is increased. It is concluded that the DNA-mediated toxicity may be more important in the hypopharynx carcinoma than in the CSM colon carcinoma. As a consequence, pre-treatment with MTX or thymidine enhances FNuc formation, although only in the CSM carcinoma is there an increased tumor growth delay. Thus, in the hypopharynx carcinoma the measurement of FNuc did not serve as a predictor for the treatment efficacy of the combined treatment modality. Pre-treatment with MTX did not influence the catabolism of 5-FU, whereas thymidine actually prolonged the half-life of 5-FU without alpha-fluoro-beta-alanine becoming detectable.

Behavior of the basement membrane during carcinoma cell invasion in chemically induced carcinomas of the skin.

Immunohistochemical localizations of the intrinsic basal lamina (BL) components laminin-1 and type IV collagen, the adhesion molecules type VII collagen and laminin receptor (alpha 6 beta 1 integrin), and the type IV collagenase (72 kDa, MMP2) were analyzed in carcinomas of the mouse skin which were chemically induced by benzo[a]pyrene with or without the addition of the promotor phorbol 12-myristate 13-acetate. Normal skin, dysplastic lesions, and invasive carcinomas were investigated by histological and immunohistochemical (immunofluorescence and APAAP) methods. A regular and continuous staining of laminin-1 and type IV collagen was present in the normal skin and in areas of slight and moderate dysplasia. Underneath highly dysplastic epithelium, the BL became thin, loosened and sometimes disrupted, and accumulations of globular BL material were found in the connective tissue underneath the BL. Type VII collagen retained a more linear, continuous and uniform distribution in the areas of progressed epithelial dysplasia. All invasive carcinomas were characterized by a BL which was disrupted by gaps of varying size but, again, showed a more uniform and less discontinuous distribution of the anchoring molecule type VII collagen. Expression of the integrin laminin receptor investigated increased quantitatively in dysplastic lesions and in areas of invasive carcinomas, showing a circular presence at the surface of most epithelial cells of the basal and spinous layers of the epidermis, whereas, in the normal skin, the laminin receptor was polarized to the basal and lateral cell membrane of basal epithelial cells in contact with the BL. These results suggest that the discontinuities occurring in the BL during carcinoma cell invasion are not caused by a local loss of the anchoring molecules type VII collagen and/or laminin receptor, though alterations in the pattern of expression of the laminin receptor document fundamental changes in the cellular organization occurring during malignant transformation. On the other hand, the presence of the type IV collagenase increased in epithelial dysplasias and invasive carcinomas. In many dysplastic lesions, it was deposited in a plaque-like and sometimes linear manner near the basement membrane (BM) region indicating that, in chemically induced carcinomas, this enzyme may be involved in the process of BM perforation during carcinoma cell invasion.

Cytokinetic effects of cisplatin on diverse human head and neck carcinomas in vitro: dependence on the tumor sensitivity to cisplatin.

Studies performed with xenografted human head and neck carcinomas in vivo have demonstrated that the cytokinetic phenomena occurring under the influence of cisplatin closely correlate with the response of the tumors to therapy. The present paper analyses whether this correlation also exists in vitro. Four human head and neck carcinoma cell lines showing different degrees of sensitivity to cisplatin, as determined by the trypan blue exclusion assay, were investigated by flow cytometry at various intervals after administration of cisplatin. Early cell-cycle blockades in the S phase always reflected a high degree of cytostatic potency of cisplatin and were usually succeeded by a pronounced inhibition of tumor cell proliferation and a reduction of cell viability. In the case of a minimal response to therapy and in untreated control cultures of all four tumor lines, the relative number of S-phase cells continuously diminished during the observation period. These findings point to the S-phase blockade as the crucial cytokinetic effect of cisplatin preceding relevant growth reductions. This knowledge might support the development of a drug-response assay that could predict the sensitivity of individual patient tumors in vitro before the beginning of clinical cancer chemotherapy.

The basement membrane and its involvement in carcinoma cell invasion.

During the development of invasive cancer, carcinoma cells have to penetrate the extracellular matrix including the basement membrane (BM). This is a usually continuous layer composed of a dense meshwork of collagens, glycoproteins and proteoglycans. It normally underlies epithelia and lacks any pores large enough to allow epithelial cells to pass through them. In consequence, the invasion of carcinoma cells through the BM must be either an active process effected by the carcinoma cells themselves or is mediated by structural alterations of the BM occurring during carcinogenesis and cancer progression. It was supposed by many authors that invading and metastasizing carcinoma cells are able to degrade actively the continuous, uninterrupted BM by secreting type IV collagenase and other proteolytic enzymes. Although there is a wealth of experimental evidence which fits the concept that the active degradation of the BM is an essential requirement for carcinoma cell invasion and metastasis, certain data do not agree with this hypothesis. Thus, it is still doubtful whether active lysis of the BM by carcinoma cells is actually a prerequisite for invasion and metastasis or whether there are alternative and/or additional mechanisms favoring the invasion of carcinoma cells.

Lack of cell-cycle-specific effects of recombinant tumor necrosis factor in vivo.

Several in vitro studies have demonstrated that tumor cells arrested in the G2 and M phases of the cell cycle expressed an increased sensitivity to the tumor necrosis factor (TNF). The scope of the present study was to investigate whether this cycle dependence of TNF effects also exists in vivo. The experiments were performed by using the Lewis lung carcinoma (LLC), which had been allotransplanted to nude mice. In order to induce delays of the tumor cell cycle in G2, the animals were treated with etoposide (40 mg/kg body weight i.p.) or with local radiation (15 Gy), each increasing the G2 fraction of the LLC from 10% to 35% and 50% respectively. For combination therapy with recombinant (r)TNF, the tumor was transplanted to four groups of six mice each, one of them serving as a control group the others being treated either with a G2 inductor alone, with rTNF alone, or with rTNF and a G2 inductor combined. Administration of rTNF (125 or 250 micrograms/kg body weight i.v.) was always carried out 24 h after therapy with etoposide or radiation when the maximum of G2 accumulation had developed. The growth behavior of the treated tumors revealed that the response of the LLC to rTNF in vivo was not improved by pretreatment with a G2 inductor and, thus, obviously lacked cell-cycle specificity. It is supposed that direct interactions of TNF with the tumor cells, which are a basic requirement for cell-cycle-linked phenomena, play a minor role in the therapeutic outcome of the LLC under in vivo conditions.

Effect of acquired cisplatin resistance on the response of a xenografted human hypopharynx carcinoma to concurrent radiochemotherapy with cisplatin.

The optimum integration of chemotherapy and irradiation is of potential clinical significance in the treatment of advanced head and neck carcinomas. In the present study, the interaction of cis-diamminedichloroplatinum(II) (cisplatin) with fractionated radiotherapy was investigated in a human hypopharynx carcinoma and a cisplatin-resistant subline of this tumor, both growing in athymic mice. Two radiochemotherapy schedules which tested single as well as combined-modality treatments were applied. After therapy, the tumor sizes were measured three times per week in order to determine growth delay and treatment:control (T/C) ratios. The results revealed approximately additive effects of both agents in the parent hypopharynx carcinoma. In the resistant subline, such an interaction did not appear after treatment with any of the investigated schedules. However, a significant cross-resistance between cisplatin and radiation was detectable. It can be concluded that multiple courses of a platinum-based induction chemotherapy may be disadvantageous, since the treated tumors may develop drug resistance which obviously limits the effectiveness of a subsequent combined-modality approach.

Complexes of metals other than platinum as antitumour agents.

The earliest reports on the therapeutic use of metals or metal-containing compounds in cancer and leukemia date from the sixteenth and nineteenth centuries. They were forgotten until the 1960s, when the anti-tumour activity of the inorganic complex cis-diammine-dichloroplatinum(II) (cisplatin) was discovered. This led to the development of other types of non-organic cytostatic drugs. Cisplatin has developed into one of the most frequently used and most effective cytostatic drugs for the treatment of solid carcinomas. Numerous other metal compounds containing platinum, other platinum metals, and even non-platinum metals were then shown to be effective against tumours in man and experimental tumours in animals. These compounds comprise main-group metallic compounds of gallium, germanium, tin, and bismuth, early-transition metal complexes of titanium, vanadium, niobium, molybdenum, and rhenium, and late-transition metal complexes of ruthenium, rhodium, iridium, platinum, copper, and gold. Several platnium complexes and four non-platnium-metal antitumour agents have so far entered early clinical trials. Gallium trinitrate and spirogermanium have already passed phase II clinical studies and have shown limited cytostatic activity against certain human carcinomas and lymphomas. The two early-transition metal complexes budotitane and titanocene dichloride have just reached the end of phase I clinical trials and have been found to have an unusual pattern of organ toxicity in man. Titanocene dichloride will soon enter phase II clinical studies.

Distribution of type-VII collagen in xenografted human carcinomas.

The distribution of type-VII collagen, the main molecular component of the anchoring fibrils (AF) attaching the basal lamina (BL, lamina densa of the basement membrane) to the surrounding connective tissue, was investigated in four xenografted human carcinomas of the hypopharynx (H-Stg 1), the lung (L 261), the sigmoid colon (CA 1), and the rectum (R 85). The studies were performed with a recently prepared, affinity-purified and highly specific antibody to type-VII collagen by using the indirect immunofluorescence and the APAAP (alkaline phosphatase anti-alkaline phosphatase) techniques. For comparison, the localization of the intrinsic BL components laminin and type-IV collagen were additionally analyzed in all four carcinomas. It was shown that type-VII collagen usually colocalized to laminin and type-IV collagen and was deposited at the borderline between carcinoma cell clusters and the surrounding strands of connective tissue in a similar, but more diffuse and less continuous distribution than both intrinsic BL components. In the squamous cell carcinoma H-Stg 1 and the adenocarcinoma L261, type-VII collagen was additionally accumulated in enlarged extracellular spaces between carcinoma cells, away from the contact zone to the connective tissue and again colocalized to laminin and type-IV collagen. Numerous carcinoma cells of both xenografts showed remarkable intracytoplasmic immunoreactivity for the antibody to type-VII collagen. Even in the case of the gastrointestinal carcinomas CA 1 and R 85, faint immunoreactivity for type-VII collagen was found at the contact zone between the mucosal epithelium and the surrounding connective tissue. These results confirm that epithelial carcinoma cells are obviously involved with the synthesis of the main molecular component of AF usually attaching the BL to the adjacent connective tissue and hint at a possible correlation between the localization of type-VII collagen and the observed pattern of the BL. However, it cannot be decided whether there is a direct causal relation between both phenomena or whether they are both the consequence of an independent but common cause, such as abnormal cellular differentiation of carcinoma cells. In no case, can the discontinuities in the distribution of type-VII collagen be explained by active tumor cell invasion since xenografted human carcinomas neither invade nor metastasize.

31P NMR spectroscopy of a xenografted hypopharynx carcinoma: effects of tumor growth and treatment with cisplatin on the tumor phosphorus metabolism, histology and cytokinetics.

A xenografted hypopharynx carcinoma growing subcutaneously in nude mice was studied by in vivo 31P NMR spectroscopy during uninfluenced growth and following treatment with cisplatin (CDDP). Parallel to the NMR experiments, the cytokinetic and histological changes in the tumor were investigated. The most significant change in the growing tumor was a decline in the level of phosphocreatine (PCr), whereas the tumor pH did not change. Following treatment with CDDP (4, 8 and 12 mg/kg), a dose-dependent decrease in the level of phosphomonoesters (PME) took place, whilst no dose dependence could be observed for the increase of PCr. the pH shifted to alkaline only after administration of the highest CDDP dose. Tumor cytokinetics revealed a cell arrest at the G1/S boundary 24 h after chemotherapy. At this time, the histological sections showed a dilatation of capillaries, whereas first necroses appeared on day 3. The proliferative activity of the tumor showed a sharp decline 24 h after CDDP application, followed by a revival of cell proliferation that was proportional to the dose applied between days 5 and 7. This increase in proliferative activity was paralleled by a marked increase in the PME/phosphodiesters ratio. Thus, in the tumor investigated the PME were the best indicators of tumor response to therapy. A precise correlation between the cytokinetic data and the re-energization of the tumor was not possible because histological changes, which may contribute to improved tumor energy status took place at the same time.

Changes in the cytoskeleton pattern of tumor cells by cisplatin in vitro.

The influence of the antitumor drug cis-diamminedichloroplatinum(II) ('cisplatin') upon the structural pattern of the main cytoskeletal components, i.e. microtubules, intermediate filaments and microfilaments, was investigated in squamous carcinoma cells derived from the mouse stomach (G 22) or the human lung (L 266) and growing in vitro as monolayer cultures. The studies were performed by the indirect immunofluorescence technique using monoclonal antibodies against alpha-tubulin, type 19 cytokeratin and actin at the end of a 90-min exposure to 2.5 x 10(-6), 5 x 10(-6) or 10(-5) mol cisplatin/l and a subsequent 24-h recovery period. Under the influence of cisplatin, the cytoskeletal tubules and filaments, which were distributed in untreated cells as a finely organized network spreading through the whole cytoplasm like a spider's web, collapsed and aggregated to dense and circularly arranged bands of bright immunofluorescence around the nucleus or to cap-like structures apposing the nucleus. These phenomena developed in clear dependence upon the dose of cisplatin applied and were observable in a modified manner and to a different degree with the three structural elements of the cytoskeleton. During the subsequent 24-h interval, during which the cells were allowed to recover in drug-free growth medium, the before-mentioned collapse of the cytoskeletal network was only partially reversible following previous treatment with the medium (5 x 10(-6) mol/l) and the high (10(-5) mol/l) dose of cisplatin and restored totally to the normal structural pattern of untreated control cells when the low dose of 2.5 x 10(-6) mol cisplatin/l had been administered before. These results give evidence that the DNA cannot be the only cellular target for the antitumor drug cisplatin, but that it also effects other intracellular lesions which cause structural alterations of cellular organelles independently of the primary molecular attack at nuclear DNA strands. Probably, these additional interactions fortify the antiproliferative effect and contribute to the achievement of important biological and cytological effects of cisplatin such as growth inhibition or giant cell formation.

An organoid culture assay (OCA) for determining the drug sensitivity of human tumors.

A model for testing chemotherapeutic agents in vitro is described. It is based on an organoid culture method which allows human carcinomas to grow in vitro and to maintain many typical in vivo properties, including 3-dimensional architecture, growth of multiple cell types, expression of morphological differentiation and formation of histotypical structures. The preservation of drug sensitivity and resistance under the conditions of our organoid culture assay (OCA) was demonstrated by investigating 3 strains of a human hypopharynx carcinoma which differed by different sensitivity to cisplatin in in vivo conditions. These differences were retained in vitro and the modified neutral-red (NR) assay was especially suitable for revealing drug-induced cytotoxic damage in OCA. On the basis of our findings, the following approach is proposed for the in vitro testing of cytostatic drugs before they are administered to patients. (1) Removal of carcinomas from patients; (2) dense cell suspensions of these carcinomas to be dropped on membrane filters at the air-medium interface, resulting in growth of solid nodules of organized carcinoma tissues; (3) addition of cytostatic drugs to the growth medium for 2 or 3 days; (4) detachment and bisection of the culture nodules; (5) determination of viable cells by NR uptake and total cell mass by the sulforhodamin B (SRB) assay; (6) determination of quotient NR:SRB absorbance, related as percentage to control value: this indicates the fraction of viable cells and gives a measure of the cytotoxic injury caused by the applied cytotoxic drug. Thus, the OCA seems to be suitable for defining the patterns of drug sensitivity and resistance of individual human carcinomas in vitro within a few days.

Antitumor activity of ionic niobocene and molybdenocene complexes in high oxidation states.

Four air-stable niobocene complexes [(C5H5)2NbCl2]+X- with X = BF4, AsF6, SbF6, SO3CF3 and the molybdenocene derivative [(C5H5)2MoCl2]2+[SbF6]2- were investigated for antitumor properties against Ehrlich ascites tumor in female CF1 mice. All compounds are new, salt-like complexes containing a cationic metallocene moiety, where the early transition metals niobium and molybdenum in the high oxidation states +5 and +6 function as central atoms. The niobocene complexes containing tetrafluoroborate (BF4-) or trifluoromethanesulfonate (CF3SO3-) as anions only induced a maximal cure rate of 50% and led to increases in life span of 182% and 178% following application of optimal doses. The other two niobocene compounds with hexafluoroarsenate (AsF6-) and hexafluoroantimonate (SbF6-) as anions and the molybdenocene derivative [(C5H5)2MoCl2]2+[SbF6]2- effected a maximal cure rate of 100% and increases in life span of 346%, 376%, and 332%, respectively, determined on the key date, i.e., on day 90 after transplantation. On applying the niobocene hexafluoroantimonate complex [(C5H5)2NbCl2] +[SbF6]-, the optimal dose range with a cure rate of 100% was rather broad and extended from 20 mg/kg to 40 mg/kg. Correspondingly, the value of the therapeutic index (TI) was high and amounted to 7.2. In the case of the niobocene hexafluoroarsenate and the molybdenocene hexafluoroantimonate complexes, the TI value decreased to 5.3 and 2.6 respectively. Neither impairments of the general condition nor any conspicuous symptoms could be detected after application of therapeutic doses of the five compounds investigated. Compared to the neutral niobocene dichloro complex [(C5H5)2NbCl2], the therapeutic range of the ionic niobocene derivative [(C5H5)2NbCl2]+[SbF6]- was broadened, the TI value markedly elevated from 2.9 to 7.2, and the toxic symptoms were impressively reduced. The niobocene hexafluoroantimonate complex was the most effective compound investigated in the present study.

Ionic rhenocene derivatives with antitumor activity.

The antitumor activity of the three air-stable bis(cyclopentadienyl)rhenium derivatives [(C5H5)2-ReCl2]+Cl-,[(C5H5)2ReCl2]+[AsF6]- , and [(C5H5)2-ReCl2]+[SbF6]- was tested against Ehrlich ascites tumor in female CF1 mice. All three compounds contain the group-7 transition metal rhenium in the +5 oxidation state as their central metal atom. They are ionic, salt-like complexes that are composed of the cationic [(C5H5)2ReCl2]+ moiety and one of the negatively charged counterions Cl-, AsF6-, or SbF6-. Both the chloro and the hexafluoroarsenate complexes induced a maximal cure rate of 100% when given either in a dose range of 120-160 mg/kg (rhenocene trichloride) or at a single dose of 180 mg/kg (hexafluoroarsenate derivative). The hexafluoroantimonate complex effected a maximal cure rate of only 50% at 60 mg/kg. For the two former compounds, the values for the therapeutic index (TI) amounted to 1.7 and 2.1, respectively. No impairment of the general condition or pathologic symptoms in the viscera could be detected by observation of the animals during the days following treatment with therapeutic doses or by autopsy of the surviving animals on the key date (day 90). The rhenocene derivatives investigated in the present study represent a new class of antitumor metallocene compounds as well as the first rhenium(V) complexes exerting cytostatic activity.

Effects of carboplatin on the testis. A histological study.

In the present study, the influence of carboplatin [diammine(cyclobutane-1,1-dicarboxylato)platinum(II)], the main and most active representative of second-generation antitumour platinum complexes, on the morphology of the testes of male CF1 mice was investigated histologically by examining semithick sections. Carboplatin was administered in doses of 30, 60, or 120 mg/kg and applied as a single intraperitoneal injection. For comparison purposes, the parent compound cisplatin [cis-diamminedichloroplatinum(II)] was administered at equitoxic doses (3, 6 or 12 mg/kg). At various intervals between days 1 and 28 after treatment, the testes were removed and embedded in Epon. Both compounds effected severe structural alterations of Sertoli cells, disrupted the blood/testis barrier, and impaired the processes both of spermatogenesis and spermiohistogenesis. The structural damage in the testes following treatment with carboplatin was at least as pronounced as that occurring under the influence of equitoxic doses of cisplatin. Within a few days, the intercellular spaces around Sertoli cells widened, the tight contacts with neighbouring cells were disrupted, the cytoplasm of Sertoli cells disintegrated and their nuclei shrank. Numerous necroses, abnormal mitotic figures of spermatogenic cells and malformed spermatozoa appeared. Severe damage was evident on days 10-21 after treatment with carboplatin, the strength of the symptoms being clearly dependent on the dose applied. The first indications of ongoing recovery processes were detected on day 21 in the case of the low dose (30 mg/kg) or on day 28 following treatment with 60 mg/kg or 120 mg/kg. These results confirm that carboplatin is at least as toxic to the testes as cisplatin and that its substitution for cisplatin in clinical therapy does not diminish the problem of drug-induced infertility following platinum-based chemotherapy.