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.

Effects of 8-chloro-cyclic adenosine monophosphate on the growth and sensitivity to doxorubicin of multidrug-resistant tumour cell lines.

We examined the in vitro effects of 8-chloro-adenosine 3':5'-monophosphate (8-Cl-cAMP), a reportedly stable, potent and site-selective analogue of cAMP, on the proliferation and sensitivity to doxorubicin (DXR) of two mouse cell lines, the B16 melanoma and Friend leukaemia, both as wild-type (B16, FLC) and DXR-resistant (B16/DXR, FLC/DXR) variants. The latter strains had characteristics of 'typical' multidrug resistance (MDR), including the over-expression of P-glycoprotein. Encouragingly, 8-Cl-cAMP affected almost equally the growth of the chemosensitive and chemoresistant variants of both cell lines. Its activity proved to be much more elevated on cells cultivated with fresh rather than heat-inactivated calf serum. In fact, the IC50 values for B16 and B16/DXR were about 4.7 microM in fresh serum and 215 microM in heat-inactivated serum; the IC50 values for FLC and FLC/DXR were about 12 microM in fresh serum and 70 microM in heat-inactivated serum. Furthermore, experiments with B16 showed that cotreatments with isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, or adenosine deaminase (ADA) greatly reduce the activity of 8-Cl-cAMP bringing it to comparable levels in fresh and heat-inactivated serum. These results indicate that the antiproliferative effects of 8-Cl-cAMP may be due principally to metabolites formed by the enzymic activities of the serum, most probably including 8-chloro-adenosine (8-Cl-adenosine), as suggested by other authors. Moreover, the dose-response curves and the IC50 values of the latter compound for the various cell lines were compatible with those observed for 8-Cl-cAMP in fresh serum. Finally, there was no evidence that 8-Cl-cAMP, either in the presence of fresh or heat-inactivated serum, or 8-Cl-adenosine may increase the sensitivity to DXR of the MDR variants of B16 melanoma and Friend leukaemia.

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.

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.

Beta-adrenergic influences on doxorubicin-sensitive or -resistant P388 leukemia cells.

Taking into account the possible regulatory influences of the beta-adrenergic system on lymphocyte proliferation as well as the proposed role of cyclic 3'-5'-adenosine monophosphate (cAMP) in the modulation of multidrug resistance (MDR) in tumour cells, we have tried to assess the status of the interactions between the beta-adrenergic system and a mouse lymphocytic leukemia, the P388, both as a doxorubicin-sensitive (P388) and -resistant (MDR) variant (P388/DXR). P388 showed a low total number of high affinity 125I-pindolol binding sites (340 +/- 33/cell, Kd 108 pM) when compared with normal splenocytes (1221 +/- 67 sites/cell, Kd 97 pM). The number of beta-adrenergic receptors was even lower in P388/DXR cells (230 +/- 41 sites/cell, Kd 101 pM). In addition, these receptors were subnormally expressed on the cell surface: only 26% and 52% of the total receptors were surface receptors in P388 and P388/DXR, respectively, whereas it was 87% in normal splenocytes. Isoproterenol slightly (less than 1-fold) stimulated cAMP accumulation in P388 and P388/DXR; the stimulation observed in splenocytes was 2.5-fold. In addition, the basal levels of cAMP appeared to be low (0.48 +/- 0.05 pmoles/10(6) cells in P388 and 0.71 +/- 0.08 pmoles/10(6) cells in P388/DXR; 3.47 +/- 0.28 pmoles/10(6) cells in splenocytes) in the two leukemias and they were only slightly (less than 2-fold) increased by forskolin, which otherwise stimulated about 15-fold cAMP accumulation in splenocytes; thus, P388 and P388/DXR were probably also defective in their adenylate cyclase activity. It can be concluded that, owing to multifactorial mechanisms, the lymphocytic leukemia P388, also as an MDR variant, is minimally sensitive to the direct influences of the beta-adrenergic system, probably including any effect of this system on drug-sensitivity.

Antioxidant defenses in a B16 melanoma line resistant to doxorubicin: an in vivo study.

A B16 melanoma line was repeatedly transplanted subcutaneously in C57BL/6 mice. On day 4 after every transplant, the animals were treated with doxorubicin (DXR), 10 mg/kg i.p. The aim of the work was to develop an in-vivo model of resistance to the antiblastic in order to analyze some possible mechanistic aspects of the process in the course of time. After 16 transplants and treatments the melanoma completely lost its sensitivity to the antiproliferative effects of maximal tolerated doses of DXR and showed over-expression of P-glycoprotein. Compared to the parental line, the in vitro resistance index was 4.6. After 27 transplants and treatments the melanoma did not increase its in vitro resistance to DXR further, and this resistance was completely reversed by verapamil. The behavior of the antioxidant defenses (superoxide dismutase, catalase, glutathione peroxidase, glutathione transferase, glutathione reductase and glutathione) was evaluated after 4, 16 and 27 transplants and treatments with DXR. At no stage did the treated melanoma show any variation in the antioxidant enzymes. Compared to the parental counterpart its glutathione levels were elevated after four treatments (+80%), when, however, the line was still sensitive to the in vivo effects of DXR, and after 16 treatments (+30%). Instead, no variation of the glutathione content was seen after 27 treatments with DXR. These results seem to exclude the possibility that the antioxidant defenses play a major role in the resistance of this B16 melanoma line to DXR. On the other hand, the low but, however, 'clinically' significant resistance of the tumor to the antiblastic seems mainly related to the mechanisms linked to the P-glycoprotein over-expression.

Modulation of the antioxidant activities in dox-sensitive and -resistant Friend leukemia cells. Effect of doxorubicin.

Tumor cell resistance to anthracyclines has been associated with increased activity against free radicals. Here, we have investigated the direct effect of doxorubicin (DOX) in the modulation of glutathione level and antioxidant activities in DOX-sensitive and-resistant cells (288 fold). The glutathione level in untreated cells was 88% greater in resistant than in sensitive cells. The activities of the superoxide dismutase, glutathione -S-transferase and glutathione reductase were respectively 24, 15 and 38% higher in resistant cells than in their sensitive counterparts. In contrast, catalase and total glutathione peroxidase were reduced in resistant cells by 18 and 21% respectively. Moreover, the activity of selenium-dependent glutathione peroxidase was lowered by 47% in the resistant as compared to the sensitive cells. Exposure of sensitive or resistant cells to low doses of DOX did not affect these levels in either cell variant. It is concluded therefore that resistance to anthracyclines may not always be associated with an elevated level of intracellular antioxidant activity enzymes.

Glutathione, glutathione S-transferases, and related redox enzymes in Adriamycin-resistant cell lines with a multidrug resistant phenotype.

Friend erythroleukemia cells (FLC) selected by exposure to Adriamycin (doxorubicin) express an approximate 2.5-fold (ARN1) or 13-fold (ARN2) resistance to the drug with various degrees of cross-resistance to other anthracyclines, vinca alkaloids, and epipodophyllotoxins. Because the redox cycling of the quinone moiety of Adriamycin is known to produce oxidative stress, however, an analysis of glutathione (GSH) and related enzyme systems was undertaken in the wild-type and selected resistant cells. In ARN1 and ARN2, superoxide dismutase (SOD) and catalase activities were slightly decreased, intracellular GSH and GSH reductase were essentially unchanged, and total GSH peroxidase, glutathione S-transferase (GST), and DT-diaphorase activities were slightly elevated. In each case there was no stoichiometric relationship between degree of resistance and level of activity. GST isozymes were purified from each cell line by HPLC GSH affinity column chromatography. Two-dimensional gel electrophoresis and western blot immunoreactivity against a battery of GST isozyme polyclonal antibodies determined that both the resistant and sensitive cells expressed isozymes of the alpha, pi, and mu classes (alternative murine nomenclature: M1, M2, M3). Of significance, both ARN1 and ARN2 cell lines expressed a unique alpha subunit which was absent from the parent FLC cell line. This isozyme presumably accounted for the increased GSH peroxidase activity (cumene hydroperoxide as substrate) found in ARN1 and ARN2 and may play a role in the small incremental resistance to melphalan found for both resistant lines. Expression of the isozyme was not stoichiometric with respect to degree of resistance. The presence of this isozyme may contribute to the resistant phenotype or may be the consequence of a more general cellular response to oxidative stress.

Cardiac peroxisomal enzymes and starvation.

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.

In vivo effects of doxorubicin and isoproterenol on reduced glutathione and H2O2 production in mouse heart.

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.