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Background: The T-cell lymphoma cell line HuT78B1, selected for resistance to Fas-mediated apoptosis, resulted unexpectedly resistant to the apoptotic and cytotoxic effects of gemcitabine (dFdC). We investigated whether this resistance was due to the impairment of the Fas/Fas-ligand (FasL) system., Materials and Methods: dFdC effects were studied in HuT78B1 and in the parental Fas-sensitive HuT78 cells exposed to inhibitors of the Fas/FasL system., Results: FasL- and Fas-blocking antibodies did not interfere with dFdC-induced apoptosis in HuT78 cells, whereas inhibitors of caspase-8, -9, -1 or -3 had partial inhibitory effects. Notably, in HuT78B1 cells there was a markedly reduced dFdC accumulation notwithstanding a high activity of the activating enzyme deoxycytidine kinase. dFdC accumulation in HuT78 cells was unaffected by a Fas-blocking antibody., Conclusion: This is the first time that the selection of a Fas-resistant cell line led to the isolation of a cell clone unable to accumulate the deoxycytidine analog dFdC. Our results show that this alteration is independent from the impairment of the Fas/FasL system.

5-Fluorouracil (5-FU) is a pyrimidine antimetabolite active against colorectal carcinoma and other malignancies of the digestive tract. Over-expression or mutation of thymidylate synthase (TS), the target enzyme of the 5-FU metabolite, 5-fluorodeoxyuridine monophosphate, is strictly correlated with cancer cell resistance to 5-FU. On this basis we investigated whether TS is a potential target for active specific immunotherapy of human colon carcinoma, which acquires resistance to 5-FU. Three TS-derived epitope peptides which fit defined amino acid consensus motifs for HLA-A2.1 binding were synthesized and investigated for their ability to induce human TS-specific cytotoxic T cell (CTL) responses in vitro. CTL lines specific for each peptide were established by stimulating peripheral blood mononuclear cells (PBMC) from an HLA-A2.1+ healthy donor with autologous dendritic cells loaded with TS peptide. Specific CTL lines showed HLA-A2.1-restricted cytotoxicity in vitro to HLA-A2.1+ target cells pulsed with the specific TS peptide and to HLA-class I matching colon carcinoma target cells over-expressing TS enzyme after exposure to 5-FU. Recognition by CTL lines suggests that these TS peptides may be potential candidates for use in a peptide-based vaccine against 5-FU resistant colon carcinoma.

Structural changes in the macromolecular targets of pharmacological agents can result in alterations in the efficacy of these agents. In previous studies Berger et al. (1) identified a variant structural form of thymidylate synthase (TS) that is associated with relative resistance to 5-fluoro-2'-deoxyuridine, in a human colonic tumor cell line. They observed that expression of the variant TS, which differs from the normal form by a tyrosine to histidine substitution at residue 33, confers a 4-fold level of drug resistance in mammalian cells, as well as in bacteria. Now we report on the use of RT-PCR techniques to see if that variant TS form could be present in human samples from patients who underwent surgery for primary colorectal cancer and been previously untreated and to try to find relationships between that hypothetical variant TS form and the 5-Fluorouracil treatment. The possible role of Tyr-33 in 5-fluoropyrimidine-mediated inhibition of TS is discussed.

Objective: To assess estrogen and progesterone receptor presence in human breast tumors using immunocytochemical analysis., Study Design: For both estrogen (ER) and progesterone (PR) receptor assay, percent of stained cells and intensity of staining were estimated on a series of 251 consecutive breast cancer cases from the M. Ascoli Cancer Hospital Center in Palermo using the CAS 200 image analysis system., Results: Cytochemical assay revealed a differential distribution of both ER and PR, by menopausal status of the patients; premenopause (PreM) was mostly ER negative (63%), and postmenopause (PostM) > 10 years was mostly ER and PR positive (64%). The percent of cells stained for ER was significantly different between PreM and PostM patients when they were considered as a whole. By contrast, no difference emerged for PR staining among menopausal groups. Overall, patients whose tumors were PR positive showed a significantly (P < .03) longer interval free of relapse., Conclusion: The present results suggest that PRs behave as better indicators than ERs of early relapse in breast cancer patients. Further studies, with longer follow-up, are needed, however, to validate this concept.

Many anticancer drugs are able to induce apoptosis in tumor cells but the mechanisms underlying this phenomenon are poorly understood. Some authors reported that the p53 tumor suppressor gene may be responsible for drug-induced apoptosis; however, chemotherapy-induced apoptosis can also be observed in p53 negative cells. Recently, doxorubicin (DXR) was reported to induce CD95L expression to mediate apoptosis through the CD95/CD95L system. Thus, an impairment of such a system may be involved in drug resistance. We evaluated the in vitro antitumor activity of several cytotoxic drugs on two human p53-negative T-cell lymphoma cell lines, the HUT78-B1 CD95L-resistant cell line and the HUT78 parental CD95L-sensitive cell line. We demostrated by Western blotting assay that DXR and etoposide (VP-16) were able to induce CD95L expression after 4 h of treatment. In contrast, they were unable to induce the expression of p53. DXR, at concentrations ranging from 0.001 - 1 microg/ml, and VP16, at concentrations ranging from 0.05 - 1 microg/ml, were equally cytotoxic and induced apoptosis in both cell lines as assessed by fluorescence microscopy and flow cytometry analyses. Although we observed a slightly reduced percentage of apoptotic cells in HUT78B1 when compared with the parental HUT78 cells after few hours of drug exposure, this difference was no longer evident at 48 or 72 h. Similarly, the exposure of HUT78 cells to a CD95-blocking antibody partially reduced early apoptosis (24 h) without affecting the long-term effects of the drugs including cytotoxicity. Furthermore, as observed with DXR and VP-16, both the CD95L-sensitive and the CD95L-resistant cell lines resulted equally sensitive to the cytotoxic effects of a number of different cytotoxic drugs (vincristine, camptothecin, 5-fluorouracil and methotrexate). The treatment with the Caspase-3 tetrapeptide aldehyde inhibitor, Ac-DEVD-CHO, did not affect the DXR-induced apoptosis whereas it only modestly inhibited apoptosis and cytotoxicity of VP-16, while Z-VAD.FMK, a Caspase inhibitor that prevents the processing of Caspase-3 to its active form, was able to block DXR-induced apoptosis at 24 h but not at 48 h. Thus, our results do not confirm a crucial role for the CD95/CD95L system in drug-induced apoptosis and suggest the involvement of alternative p53-independent pathways at least in this experimental model system.

Inhibition of Thymidylate Synthase (TS) by the 5-Fluorouracil (5Fu) active metabolite Fluoro-deoxy-uridine-monophosphate (FdUMP) is considered to be the main mechanism of action of 5Fu. TS level from tumors and normal mucosa of 62 untreated patients who underwent surgery for primary colorectal adenocarcinoma was performed. The aim of this study was to evaluate the possibility of considering the TS level as a prognostic factor of the disease. A large variation in the level of the enzyme was found among tumors. Our data demonstrate that there is no association with age, sex, and tumor size; however there are significant relationships between TS levels and staging and histological grading. In fact the TS values are higher in Dukes' A and in G1 than in Dukes' D and G3 tumors (p < 0.05). Another significant association has been found between the TS level and tumor site: pts with right colon neoplasias had higher TS levels than pts with left and rectum ones. An interesting trend was found between the TS levels and survival parameters. Pts who had lower TS levels had a significantly increased risk of death (p < 0.05) over pts with a higher outcome. Our data support the hypothesis that a high TS level is a favourable prognostic factor in human untreated colorectal carcinomas according to our previous preliminary data (1).

5-Fluorouracil is the drug chosen for the treatment of patients with advanced colorectal carcinoma; its major site of action is thymidylate synthase (TS), resulting in pronounced and prolonged inhibition of DNA synthesis. The aim of this study was to evaluate the possibility of considering TS level in human colorectal carcinomas of previously untreated patients (pts) as a prognostic factor. Our data demonstrate that there is no association with age, sex, tumor site and tumor size; however, there is a relationship between TS levels and staging: in fact, the TS values are higher (P < 0.05) in Dukes-A tumors than in the others. A significant association was also found between the TS levels and survival parameters: in fact, pts with longer disease-free and overall survivals had a significantly increased TS level compared to pts with a poorer outcome (P < 0.05). Moreover, pts with DNA-aneuploid tumors had lower TS level (median = 0.044 pmol/mg protein) than diploid pts who had higher TS level (median = 0.093 pmol/mg protein); however the difference is not significant. Our result are based on preliminary data; however, they seem to support the hypothesis that a high TS level is a favourable prognostic factor in human colorectal carcinoma.

In this paper we report the results obtained treating a multidrug resistant (MDR) murine erythroleukemia cell line with daunomycin (DNM) in association with two new modulators characterized by a favourable therapeutic index, lacidipine (LCD), a dihydropyridine calcium antagonist, and josamycin (JSM), a macrolide antibiotic. LCD and JSM exhibited a greater MDR reversal activity than verapamil (VRP) and erythromycin (ERY) respectively. The accumulation of DNM in the DRTL cells exposed to modulators was similar to that of the parental cell line FLC. In the case of LCD, it was possible to ascertain that at a very low concentration this molecule can circumvent MDR without modifying DNM accumulation, suggesting that multiple different determinants may be responsible for MDR other than P-170 in this cell line.

CD1 female mice were treated with doxorubicin (DXR, 5 mg/Kg i.v.) once a week for eight weeks or with lonidamine (LND, 100 mg/Kg i.p.) once a week for eight weeks. Other mice received lonidamine (100 mg/Kg i.p.) immediately before doxorubicin (5 mg/Kg i.v.) administration. The animals were sacrificed four weeks after the last administration and their cardiac morphology was evaluated by means of light microscopy. Lonidamine slightly reduced the extent of the atrial but not of the ventricular alterations caused by DXR. The results seem to indicate that, in this experimental model, lonidamine does not substantially modify the cardiotoxicity induced by doxorubicin.

Biological agents might offer various therapeutic opportunities in the treatment of cancer, including a direct and/or host-mediated antiproliferative effect and also the possibility to favorably modulate tumor resistance to antineoplastic drugs. We studied the in vitro antiproliferative effects of interferon (IFN)-gamma on the mouse B16 melanoma and Friend erythroleukemia, and the human K562 erythroleukemia, as doxorubicin (DXR)-sensitive and -resistant (multidrug resistant) variants. These effects were marked in B16 melanoma and rather slight in K562 erythroleukemia, without any difference between the DXR-sensitive and -resistant lines. The chemosensitive variant of Friend erythroleukemia showed an intermediate response, which was greater than that seen in its resistant counterpart. There was no apparent relationship between the antiproliferative activity of IFN-gamma and the glutathione content of the cell lines. On the other hand, this activity was enhanced by co-treatment with glutathione-depleting concentrations of buthionine sulfoximine, but only in the cell lines which had responded better to IFN-gamma alone. This result probably confirms that a free radical mechanism plays a part in the antitumor effect of the cytokine. Finally, a range of concentrations of IFN-gamma, including slightly cytotoxic ones, did not substantially improve the antiproliferative effects of doxorubicin on the various cell lines, except in the DXR-sensitive variant of Friend erythroleukemia where a synergistic effect of the combination was observed. Thus, our results are not very promising with regard to a possible favorable modulatory activity by IFN-gamma of DXR (multidrug)-resistance.

Some 3-triazenopyrroles were prepared by coupling 3-diazopyrroles and secondary amines. In preliminary in vitro screening tests derivatives 3 showed to be cytotoxic and exhibited mutagenic effects.

After informed consent 21 patients with advanced head and neck cancer resistant to folinic acid/5-fluorouracil (FA/5FU + cisplatin) were treated with weekly FA/5FU plus low dose hydroxyurea (HU) to evaluate if HU could further modulate 5FU antineoplastic activity. Five patients achieved a partial response (23.8%) which was short-lived (mean duration 6.5 months). Three patients (14%) had stable disease and 13 (62%) progressed. Among responders, four patients had epidermoidal carcinoma and one had clear cell carcinoma. Treatment was well tolerated and 5FU-related toxicity was not apparently worsened by the addition of HU. The most frequent toxicities were nausea/vomiting (81%), diarrhea (52%) and leukopenia (57%). Grade 3 nausea/vomiting and leukopenia were recorded in only 19 and 9% of cases, respectively. One patient had grade 1 cutaneous toxicity and a second patient showed a hand-foot syndrome. These results suggest that HU may further positively modulate 5FU antineoplastic activity.

Background: Resistance of tumor cells to cytotoxic agents can be due to the overexpression of the mdr 1 gene, which encodes a plasma membrane protein (P-glycoprotein). To understand the molecular basis of multidrug resistance, several laboratories have isolated cell lines resistant to doxorubicin, actinomycin D, vinca alkaloids and related agents. Many months or years of culture with gradually increasing concentrations of cytotoxic agents are necessary to obtain a resistant cell line., Methods: We selected a new multidrug resistant cell line (MELC-DRTL) by 24-hour cycles of exposure to relatively high concentrations of daunorubicin from sensitive Friend Leukemia cells. After each cycle, the residual live cells were expanded up to the density of 1 x 10(6) cells/ml., Results: The assay conducted with MoAb C-219 showed a high expression on the membrane surface of P-glycoprotein in the MELC-DRTL line, but the fact that it was impossible to obtain a complete reversal of the resistance, even when using high concentrations of verapamil, suggests the presence of other mechanisms unrelated to the presence of P-glycoprotein., Conclusions: The kind of cellular resistance induction used in this experiment enabled us to obtain an MDR cell line in three months of culture.

Recent studies suggest that aspartic proteinase Cathepsin D may be implicated in the process of tumor invasion and metastasis. In fact several in vitro observations showed that this proteinase may facilitate the spread of neoplastic cells through different mechanisms related to its proteolytic activity, by acting at different levels of the metastatic cascade. Cathepsin D may promote tumor cell proliferation by acting as an autocrine mitogen through the activation of latent forms of growth factors or by interacting with growth factor receptors. The enzyme was also shown to be able to degrade in vitro extracellular matrix and to activate latent precursors forms of other proteinases involved in the invasive steps of the metastatic process. Although unequivocal proof of its active role in promoting these processes also in vivo has not been obtained so far, recent clinical observations which showed a positive correlation between levels of expression of Cathepsin D activity and malignant progression of some human neoplasms further support this hypothesis. These findings warrant extensive experimental and clinical studies to better assess the pathophysiological role of this acid proteinase in the spread of neoplastic diseases and suggest new and more selective therapeutic approaches to the treatment of human neoplasms.

A phase II trial of citrovorum factor, 500 mg/m2/week, plus 5-fluorouracil, 400 mg/m2/week on day 1, and cisplatin, 20 mg/m2/week on day 2, was carried out in a group of 40 patients with metastatic colorectal carcinoma. A partial response with a mean duration of 8.4+ months was achieved in 24% of patients, a minimal response with a mean duration of 5.4 months was obtained in 6% of patients, and a stabilization of 6.2 months was achieved in 41%. Ten patients (29%) progressed. A 38% partial response rate was seen in patients with advanced rectal carcinoma, whereas no response was obtained in patients with colon cancer. Interestingly, 5 partial responses were seen in 12 patients pretreated with 5-fluorouracil. The overall survival was 9.8+ months. The mean survival of patients who achieved a partial response was 12.0+ months, whereas patients who progressed survived a mean of 6.6+ months. Patients with colon cancer had a mean survival of 8.1+ months, and those with rectal cancer survived a mean of 11.1+ months. This difference was not statistically significant. The treatment was generally very well tolerated, with patients showing mostly grade 1-2 gastrointestinal and/or hematological toxicity.

A group of 60 patients with advanced head/neck cancer were treated with high-dose folinic acid (500 mg/m-2/week-1) plus 5-fluorouracil (400 mg/m-2/week-1 on day 1, and cisplatin (20 mg/m-2/week-1) 24 h after folinic acid infusion was completed. Out of 55 evaluable patients, 10 patients (18%) experienced a complete response with a mean duration of 11.4+ months, 25 patients had a partial response (45%) of 6.7+ months, 6 patients (11%) showed a stabilization of 4.8+ months, and 14 (25%) progressed. The overall response rate was 63.6% (95% confidence limits 56.5%-69.5%). Patients pretreated with radiotherapy had a 67% overall response rate, while those pretreated with chemotherapy showed a 54% overall response rate. All patients with cancer of the oropharynx had a major response, while patients with cancer of the oral cavity had the lowest response rate. The mean survival of patients who attained a complete response was 14.5+ months. Partial responders had a mean survival of 10.6+ months, while patients who progresses survived a mean of 3.6+ months. The treatment has been very well tolerated with few cases of grade 3 gastrointestinal toxicity. Grade 1-2 leukopenia was recorded in 64% of cases, grade 1-2 nausea/vomiting in 85%. In one case therapy was stopped because of persistent diarrhoea.

Twenty-three cancer patients with pleural and/or abdominal effusion refractory to chemotherapy were treated with intracavitary beta-interferon after drainage. The overall response rate at 30 days was 35% for the whole group of patients, 25% for those with pleural effusion, and 40% for those with ascites. In 15 patients the mean time to re-accumulation of fluid after drainage only was 8.7 days, while it approached 30.3 days after intracavitary beta-interferon. This difference was statistically significant (P less than 0.001). The treatment was well tolerated with only mild and transient pain in 3 cases.

In view of the structural similarity of mitoxantrone to anthracyclines and its ability to intercalate into DNA, we studied its influence on the synthetic processes of DNA and proteins in CD-1 mice tissues. By studying at the DNA level the impairment of 3H-thymidine incorporation and its return to normal, it was found that bone marrow and spleen showed similar behavior, i.e., a rapid return to normal, which occurred before bone marrow cell number and spleen weight returned to basal values. At the cardiac level, the incorporation values of precursors into DNA, reduced by treatment with mitoxantrone, came back very slowly to the control ones. Hepatic DNA showed a lower sensitivity to mitoxantrone. Analysis of 3H-leucine incorporation into three protein fractions of the heart showed that the contractile proteins were the most responsive fractions to mitoxantrone treatment. Experiments on CD-1 mice treated repeatedly with mitoxantrone revealed that the antitumor drug, at the cumulative dose of 8 mg/kg i.v., induced alterations in myocardiac morphology similar qualitatively to those induced by doxorubicin, although smaller quantitatively.

Recombinant human erythropoietin given subcutaneously is able to accelerate hematopoietic recovery in hamsters affected by cisplatin-induced anemia, increasing hemoglobin and hematocrit levels. The increase in hemoglobin and hematocrit levels. The increase in hemoglobin and hematocrit levels is not accompanied by an increase of platelet or granulocyte counts in the peripheral blood. However, an increase in the marrow concentration of burst and colony forming units--erythroid (BFU-E, CFU-E) is observed after recombinant human erythropoietin treatment. This increase in the concentration of marrow erythroid progenitors was accompanied by an increase in the percentage of these stem cells in DNA synthesis as assessed by exposure to ARA-Cyt. Recombinant human erythropoietin seems highly effective in ameliorating and/or preventing cisplatin-induced anemia in experimental animals.

A combination of mitomycin C, vindesine and melphalan was administered to 33 patients with heavily pretreated refractory breast cancer. The overall response rate was 27% with a mean duration of more than 10.2 months. A stabilization with a mean duration of 5.1 months was seen in 56% of cases, while 20% of patients progressed. Gastrointestinal toxicity, mostly grade 1 or 2 nausea/vomiting was seen in 85% of cases, grade 1 or 2 leukopenia in 60% of patients, and grade 1 or 2 thrombocytopenia in 42%. Considering the good compliance of this regimen and the poor prognosis of patients with refractory advanced breast cancer, this combination can be useful as a palliative treatment of breast carcinoma.

CD 1 female mice were treated with Doxorubicin (5 mg/Kg i.v.) once a week for 8 weeks or with Isoproterenol (20 mg/Kg s.c.) once a week for 5 weeks. Other mice were treated with the chelating agent ICRF-187 (100 mg/Kg i.p.) 30 min. before Doxorubicin or Isoproterenol administration. The animals were sacrificed 4 weeks after the last administration and their cardiac morphology was evaluated by means of light microscopy. ICRF-187 significantly lessened the extent and the severity of the cardiac lesions by Doxorubicin (-68%, P less than 0.01 in left atrium; -69%, P less than 0.01 in ventricles) and the extent of those induced by Isoproterenol (-56%, P less than 0.05). These data confirm that ICRF-187 has good activity on Doxorubicin-induced myocardiopathy and provide new information about the "in vivo" effects of the compound on the cardiotoxicity caused by Isoproterenol. Moreover, they seem to confirm that a common event, probably the involvement of metal ions, plays a role in the morphologically different myocardiopathies induced by Doxorubicin or Isoproterenol.

An in vitro study was carried out to evaluate the possible protective effect of recombinant human erythropoietin (rhEpo) on cisplatin (CDDP)-induced inhibition of erythroid colony growth. CDDP at the dose of 0.4 mg/ml caused a 72-75% inhibition of erythroid stem cell growth in plasma clots. In the short-term assay rhEpo was not able to protect normal murine erythroid stem cells from the inhibitory effect of CDDP at any of the tested concentrations. In long-term bone marrow cultures, although the kinetics of cellular populations in suspensions and of erythroid stem cells were similar in both controls and CDDP-treated cultures, both cellularity and erythroid stem cells levels were much lower in CDDP-treated flasks than in control ones regardless of the addition of rhEpo. These results indicate that rhEpo is not able to prevent of ameliorate erythroid stem cell inhibition due to a possible toxic effect of CDDP, at least in vitro.

Thirteen patients with histologically proven advanced and/or metastatic renal cell carcinoma (RCC) were treated with vinblastine (Velbe, Eli Lilly, Sesto Fiorentino, Italy) and interferon-alpha-2a (Roferon-A, Roche, Milan). Eleven out of 13 patients were evaluable. Eighteen percent of patients had partial response, 46% stable disease (SD), and 36% progressive disease (PD). The mean survival of responders was 228+ days, whereas the patients showing SD and PD had a mean survival of 154+ and 107+ days respectively. Toxicity, including influenza-like syndrome, fever, neurological and gastrointestinal side effects, was generally mild. However, medication with paracetamol was required in 82% of cases. Our small study confirms that VBL and IFN-2a regimen is moderately active in RCC.

In previous studies we reported that teniposide (VM26) induced acute cardiac effects in dogs seem to be related to a release of histamine and that a prior treatment with chlorpheniramine, an H1 histamine blocker, prevents the onset of this phenomenon. Since histamine and other vasoactive substances also seem to be involved in doxorubicin (DXR)-induced acute cardiac effects, experiments were undertaken in the aim to prevent, as in the case of VM26, the onset of this phenomenon by administering chlorpheniramine. Since DXR-induced chronic cardiomyopathy also seems to be related to the same mechanisms involved in the onset of acute cardiac effects induced by this drug, additional studies were carried out to investigate whether a long-term treatment with VM26 could induce in mouse alterations of cardiac morphology similar to those of DXR. In addition, because the mouse is known to be extremely insensitive to histamine, further studies were performed to investigate whether DXR or VM26 administration could induce in this animal model a massive histamine release and whether a long-term treatment with high doses of histamine could elicit, similarly to DXR, alterations in cardiac morphology. The results of our experiments demonstrated that DXR (1.5 mg/kg i.v.) caused in the dog a massive histamine release and a marked impairment of cardiac inotropism. As previously described for VM26, prior treatments with chlorpheniramine completely prevented this phenomenon. Furthermore, DXR administration, at a dose level able to induce cardiac damage in the mouse (2.5 mg/kg i.v.), or that of VM26 (2 mg/kg i.v.) failed to induce a massive histamine release. In addition, long-term treatment with VM26 (2 mg/kg i.v.) or high doses of histamine (100 mg/kg i.v.), unlike DXR, did not elicit in this animal alterations of cardiac morphology. Finally, chlorpheniramine (0.15 or 0.45 mg/kg i.v.) did not prevent the onset of chronic cardiomyopathy induced by DXR in mouse. In conclusion, our results show that the role of histamine in the onset of DXR-induced chronic cardiomyopathy, at least in mouse, remains questionable and suggest that this animal, because of its high natural resistance to histamine, is not a suitable experimental model to investigate the cardiovascular pharmacology of drug-induced histamine release.

A group of 23 consecutive patients with biopsy-proven advanced or metastatic head and neck cancer were treated with cisplatinum, 100 mg/m2 i.v., on day 1 plus 5-fluorouracil, 1000 mg/m2, in continuous infusion for 5 days. Most patients (87%) had recurrent or metastatic cancer and were previously treated (78%). Out of 21 evaluable patients we obtained a 42% overall response rate (complete + partial responses) with a mean duration of more than 8 months and a 14% minimal response rate. A stabilization of disease was achieved in 28% of cases, while 14% of patients progressed. This response rate, as well as the duration of response, seems to be similar to those obtained in other series comprising previously treated patients with advanced or metastatic head and neck carcinoma. The toxicity was generally acceptable, with few cases of grade 3 (WHO criteria) toxicity. However most patients required hospitalization because of the length of treatment. In conclusion the response rate and the duration of responses obtained with cisplatinum plus a 5-day infusion of 5-FU in advanced or metastatic pretreated patients is, at present, unsatisfactory, even if the impact on survival is still not entirely clear.

1. We have investigated the kinetics of inhibition of cathepsin D in heart, liver and skeletal muscle of CD-1 mice following administration of 25, 50, 100 and 200 mg/kg i.p. of pepstatin A, a specific inhibitor of this protease. 2. In the liver, a significant inhibition of cathepsin D occurred up to at least 15 days, whereas, in heart and skeletal muscle, this inhibition lasted for a much shorter period of time. 3. These results show that the recovery of enzyme activity to normal values is dose-dependent and that, at the same dose level, marked differences occur in the recovery of enzyme activity in these organ tissues, the liver being the most sensitive one.

The cardiac morphology of CD 1 mice undergoing two different schedules of acute (5 day) starvation and that of animals treated with a single dose (15 mg/kg i.p.) of doxorubicin, epirubicin or mitoxantrone were studied by light microscopy. Determinations of heart catalase were also carried out. Mice subjected to moderate starvation had a mean weight reduction of 18.7% and did not show heart morphological damage. A slight increase (38%) of heart catalase specific activity occurred in these animals. In animals subjected to severe starvation the weight loss was 32.2%. In this case considerable heart damage, in the form of myofibrillar loss, and a striking increase of catalase (158.5%) were seen. In the drug groups comparable weight reductions (about 15%) occurred 5 days after the treatment. Moderate heart lesions, represented by myolysis and especially by myocytic microvacuolation, were observed and appeared to be of similar degree in the 3 drug groups. Catalase specific activity increased by 119.9% in the doxorubicin animals, by 73% in the epirubicin mice and by 30.3% in the mitoxantrone ones. Light microscopy made it possible to distinguish between cardiac alterations induced by starvation and those specifically induced by antiblastics. Catalase may be helpful to indicate the existence of heart damage but it does not correlate well with the severity of the lesions by antiblastics. An additional cause of heart catalase elevation might be the free radical generation induced by the anthracyclines but not by mitoxantrone.

Some clinical studies that were performed for the purpose of assessing the potential cardiotoxicity of mitoxantrone (DHAD) have shown that repeated administrations of the drugs in some patients cause a mild impairment of cardiac functions and morphological changes in the myocardial cells qualitatively similar to those elicited by anthracyclines. Since doxorubicin has been reported to cause acute cardiac effects, probably related to its chronic cardiotoxicity, experiments were carried out on the rabbit heart to investigate whether DHAD is also able to induce acute cardiac effects. Our results show that this drug caused a reversible dose-related impairment of cardiac contractility on the isolated and perfused rabbit heart while it was not able to induce ECG alterations in normal rabbits. These findings demonstrate that in the rabbit DHAD induces an acute cardiac activity qualitatively similar to that of doxorubicin and suggest that this animal model could be a useful tool to study the cardiac actions and related pathogenetic mechanism(s) of this antitumor drug.

To correlate animal experiments with clinical experience, CD-1 mice were treated weekly for eight weeks with 4 mg/kg doxorubicin or 2.7 mg/kg 4'-deoxy-doxorubicin i.v. Periodically the incorporation of 3H-thymidine (3H-dThd) into DNA was measured in the heart, liver, spleen and bone marrow, 24 or 72 hours after the last treatment. The morphology of the hearts was examined by light microscopy. In animals sacrificed 24 hours after the last treatment, the incorporation of 3H-dThd was reduced considerably and to a similar extent by both molecules in the first weeks, while at the 8th week the values for the heart were higher than in the controls. In the other organs, inhibition percentages of incorporation of 3H-dThd into DNA persisted for both molecules, with higher values in the spleen. In animals killed 72 hours after the last treatment, no change in incorporation capacity was found. These results point to the role of cardiac DNA repair in understanding the pharmacodynamics of these two molecules. The data are discussed in relation to morphology.

Some parameters of free radical generation were studied in the hearts of CD 1 mice at short time intervals after the administration of Doxorubicin (15 mg/Kg i.v.) or Isoproterenol (80 mg/Kg s.c.). The two drugs consistently caused a decrease in cardiac reduced glutathione. Isoproterenol significantly increased in vivo H2O2 production by the heart at all the intervals taken into consideration, i.e., 5, 8, 12 and 16 hours after its administration. However, Doxorubicin did not significantly modify H2O2 generation at the same hours. These findings suggest that an increase in H2O2 production may not be involved in the oxidative stress caused by the anthracyclines at cardiac level.

Experiments in mice treated with a single 10 mg/kg dose of doxorubicin (adriamycin) i.p. revealed considerable reduction in the incorporation of 3H-thymidine in DNA and of 3H-uridine in RNA, in the spleen and liver, and at mitochondrial and non-mitochondrial level in the heart. Although protein syntheses in the heart and spleen were not reduced by the drug to any great degree, they took 10 days to return to normal; conversely, liver protein syntheses were not inhibited at all and indeed presented signs of stimulation.

The behaviour of heart and liver catalase was studied 4 days after the administration of different doxorubicin doses to CD 1 mice. The antiblastic increased the specific activity of the heart enzyme with a clear dose-response relationship (+27% after 5 mg/kg i.p.; +61% after 7 mg/kg; + 108% after 10 mg/kg; + 147% after 15 mg/kg). This did not occur in the liver where, on the contrary, a significant reduction of catalase (-31%) was noticed after the highest dose. Analyses by gel filtration excluded the possibility that doxorubicin induces major changes in the molecular properties of heart catalase. In vivo experiments with aminotriazole, which blocks catalase irreversibly, indicated that doxorubicin stimulates the synthesis of cardiac catalase. These findings are discussed with reference to the possible mechanisms of anthracycline cardiotoxicity. The catalase elevation could represent a reaction by the heart to free radicals generated by doxorubicin.

Catalase activity was followed up in the hearts and livers of CD 1 mice treated with Doxorubicin 4 mg/Kg, i.v., weekly for 9 weeks. In this murine model the antiblastic induces cardiac morphological lesions which are progressively severer with the increase of the administered cumulative dose. Heart catalase showed a consistent elevation which reached a maximum (+116.2%, P less than 0.05) after the 5th dose. In the case of hepatic catalase no significant variation was observed except a transitory elevation following the first administration. The specific increase of heart catalase activity following multiple Doxorubicin doses could be an indicator that an enhanced free radical generation acts "in vivo" along with the onset of the cardiac lesions due to antiblastic.

In mice subjected to 3-day periods of food deprivation an increase in plasma free fatty acids occurred together with a rise in the cardiac content of fatty acyl CoA-oxidase (+ 15.2%) and catalase (+ 136.2%) activities. Stimulation of hydrogen peroxide production by the heart was found after 30 hours of fasting and this phenomenon was almost completely eliminated by 6 hours of refeeding. These data suggest that high myocardial loads of free fatty acids involve the peroxisomal enzymes in the beta-oxidation process. The resulting increase in hydrogen peroxide production could be partly responsible for the myocardial injury caused by starvation.