Development and partial characterization of a human T-lymphoblastic leukemic (CCRF-CEM) cell line resistant to etoposide. Analysis of possible circumventing approaches.

We have selected an etoposide-resistant variant (CCRF-CEM/VP-16) of the human T-lymphoblastic CCRF-CEM leukemia for study. Resistance to the topoisomerase II (topo II) inhibitor was about 11-fold and stable. Other data revealed that the new cell line had acquired an atypical, non-P-glycoprotein overexpressing multidrug resistant (MDR) phenotype with cross-resistance to other topo II inhibitors (amsacrine, doxorubicin, and mitoxantrone) and to glucocorticoids, but not to novobiocin, ICRF-187, vincristine or cisplatin. In a first instance, we assumed that altered drug-topo II interactions, based on quantitative and/or qualitative modifications of the enzyme, are a cause of resistance in the cell line. We tried to modify the drug sensitivity of the cells by means of various agents and cytokines. Positive results were obtained with verapamil and, to a lesser extent, cyclosporin A, but they were not specific for the drug resistant variant and occurred in the parental CCRF-CEM as well. Other attempts with buthionine sulfoximine, novobiocin, pentoxifylline, interleukin-1, interferon-alpha, retinoic acid, TNF-alpha, bryostatin 1 or phorbol myristate acetate were substantially unsuccessful, thus confirming the difficulty of pharmacologically overcoming atypical MDR. More encouragingly, however, CCRF-CEM/VP-16 cells exhibited hypersensitivity to other agents, including actinomycin D and taxol.

Cathepsin D, B and L circulating levels as prognostic markers of malignant progression.

Growing evidence indicates that lysosomal Cathepsins D (CD), B (CB) and L (CL) may promote carcinogenesis and tumor progression. Therefore, we evaluated their potential value as biochemical parameters of malignant progression in patients with benign diseases which may undergo malignant transformation, such as liver cirrhosis (LC) and chronic pancreatitis (CHP) as well as in hepatocellular carcinoma (HCC) and pancreatic cancer (DPC). CD, CB and CL serum levels were determined by immunoenzymatic assays in LC, CHP, HCC or DPC patients and correlated with a number of biochemical and clinical parameters of these diseases. CD serum levels were increased in LC, CHP and HCC, but not in the DPC group as compared to normal subjects (NS) (P < 0.01). Interestingly, higher levels of this enzyme were observed in LC patients compared to HCC patients ( P < 0.01). CB serum concentrations were increased in all patient groups (P < 0.01). However no difference was evidenced between benign and malignant diseases. CL serum levels were significantly increased only in DPC as compared to NS (P < 0.01) or CHP patients (P < 0.02) and in HCC as compared to NS (P < 0.01). The evaluation of CD, CB and CL serum pattern in LC, CHP, HCC and DPC patients may be useful as additional biochemical parameters in the differential diagnosis and therapeutic monitoring of these diseases. Prospective clinical investigations to assess the potential value of these enzymes as biochemical markers of malignant progression of LC or CHP are warranted by the present data.

Combined activity of interleukin-1 alpha or TNF-alpha and doxorubicin on multidrug resistant cell lines: evidence that TNF and DXR have synergistic antitumor and differentiation-inducing effects.

We report on the antiproliferative effects that interleukin-1 alpha (IL-1) or TNF-alpha (TNF) in combination with doxorubicin (DXR) exert on DXR-sensitive (B16 melanoma, Friend, K562 and CCRF/CEM leukemias) and -resistant (B16-DXR, FLC-DXR, K562-DXR) cell lines in vitro. Multidrug resistance (MDR) of the latter lines entails cross-resistance to vincristine and overexpression of P-glycoprotein. Il-1 showed only a very marginal growth inhibitory activity and the effects of its combination with DXR were essentially additive in all the cell lines, except in chemosensitive B16, where a slight synergism occurred. TNF demonstrated greater antiproliferative activity in the MDR B16 and Friend tumors than in their parent variants. The combination of TNF and DXR produced synergistic growth inhibition in B16, K562 and, particularly, also in the MDR sublines of these two tumors. In addition, TNF and DXR induced synergistically erythroid differentiation in K562 and multidirectional differentiation in K562-DXR. The synergism was critically schedule-dependent in that it was achieved only when DXR application preceded or was simultaneous with that of TNF. Finally, TNF did not modify drug accumulation and retention in the cells. Our present findings stress especially the fact that DXR and TNF may exert useful antitumor synergism even in MDR lines; however, it is not likely that their interaction will occur at the specific MDR process level.

Effect of buthionine sulfoximine on the sensitivity to doxorubicin of parent and MDR tumor cell lines.

We have studied the interaction of glutathione-depleting concentrations of buthionine sulfoximine (BSO) with the anti-proliferative activity of doxorubicin (DXR) in three tumor lines, the mouse B16 melanoma. Friend erythroleukemia and the human K562 leukemia, both as DXR-sensitive and-resistant (with typical multidrug resistance) variants. BSO significantly enhanced the DXR effects in the wild-type Friend and K562 leukemias, and especially in the drug-resistant subline of Friend leukemia. BSO did not modify DXR accumulation and retention in the latter clone. Moreover, neither BSO nor verapamil used alone completely reversed the resistance to DXR of this cell line; their combination was more efficient and increased its drug sensitivity to a level closer to that of the parental counterpart. These results seem to indicate that the status of glutathione and of the enzymes related to it contributes to the resistance of Friend leukemia to DXR. An interesting additional finding was that BSO significantly synergizes with the antiproliferative effects of vincristine in the drug-sensitive variants of Friend and K562 leukemias.

Combinative growth-inhibitory effects of interferon-alpha and Doxorubicin on multidrug-resistant tumor-cell lines.

The antiproliferative effects of interferon-alpha (IFN-alpha) and of its combination with doxorubicin (DXR) were studied on three tumor cell lines, the mouse B16 melanoma and Friend erythroleukemia and the human K562 erythroleukemia, both as DXR-sensitive (B16, FLC, K562) and resistant (B16-DXR, FLC-DXR, K562-DXR) variants; the latter lines were endowed with typical multidrug resistance (MDR). Growth inhibition by IFN-alpha was greater in B16-DXR and FLC-DXR than in their chemosensitive counterparts and was superimposable in K562 and K562-DXR. On the other hand, the combination of IFN-alpha and DXR turned out to be merely additive in B16, B16-DXR, K562 and K562-DXR; it was synergistic in FLC and antagonistic in FLC-DXR. Our results and those of others seem to indicate that synergy between IFN-alpha and DXR may occur rarely in MDR cells.

Morphological changes in the hearts of CD1 mice following chronic treatment with doxorubicin and lonidamine.

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.

Antiproliferative and chemomodulatory effects of interferon-gamma on doxorubicin-sensitive and -resistant tumor cell lines.

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.

Ameliorative effects of ICRF-187 [(+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane] on the cardiotoxicity induced by doxorubicin or by isoproterenol in the mouse.

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.

Morphological changes and catalase activity in the hearts of CD 1 mice following acute starvation or single doses of doxorubicin, epirubicin or mitoxantrone.

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.

The role of histamine in doxorubicin and teniposide-induced cardiotoxicity in dog and mouse.

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.

Effects of multiple doxorubicin doses on mouse cardiac and hepatic catalase.

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.

The influence of multiple treatment with doxorubicin or 4'-deoxy-doxorubicin on the DNA synthesis and morphology of mouse heart.

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.

Effects of amsacrine (m-AMSA), a new aminoacridine antitumor drug, on the rabbit heart.

There is emerging clinical evidence that amsacrine (m-AMSA) administration may be associated with cardiotoxic effects such as severe, even fatal, ventricular arrhythmias and impairment of the inotropic performance of the heart. Information on the cardiac effects of m-AMSA in animals is scanty. Studies on mice, dogs, and monkeys have not evidenced the cardiotoxicity of the compound. The data presented in this paper show that m-AMSA causes acute ECG alterations in normal rabbits and a dose-related negative inotropic effect on the isolated rabbit heart, suggesting that this species may be a useful model for the study of the cardiac actions of this antiblastic.