The effect of ethyldeshydroxy-sparsomycin and cisplatin on the intracellular glutathione level and glutathione S-transferase activity.

Ethyldeshydroxy-sparsomycin (EdSm) is a ribosomal protein synthesis inhibitor which synergistically enhances the antitumor activity of cisplatin against L1210 leukemia in vivo. Because cellular glutathione (GSH) and glutathione S-transferases (GST) are reported to interfere with the antitumor activity of cisplatin, we analyzed the effect of EdSm and cisplatin on GSH and GST activity in selected tumor cells. For this purpose we used three murine leukemia tumors with different sensitivities towards EdSm and cisplatin: L1210-WT, sensitive to both drugs, L1210-Sm, resistant to EdSm, and L1210-CDDP, resistant to cisplatin. No significant differences were detectable between these three cell lines regarding the population doubling time, the cell size, and the cellular level of protein and glutathione. Neither of the resistant L1210 subclones showed P-glycoprotein expression. Drug exposure, however, changed the intracellular dynamics. Exposure to EdSm strongly decreased the amount of cellular protein, decreased the overall GST activity and led to GSH depletion, whereas exposure to cisplatin induced a rise in the amount of protein, in GSH, and in the total GST activity. These effects are dose-dependent and correlate well with the sensitivity of the tumor cells for EdSm or cisplatin. In addition, exposure to EdSm lowered the V(max) of GST in L1210-WT and L1210-Sm; however, in L1210-CDDP both the V(max) and the K(m) were increased. That this was not a direct effect of EdSm on GST was shown in a cell-free system, where EdSm did not influence the GST activity nor could it act as a substrate for GST. Our results suggest that the synergistic combination of EdSm and cisplatin might be explained by EdSm switching off the cellular detoxification mechanism for cisplatin, i.e. by inhibition of de novo synthesis and subsequent depletion of GSH and GST.

Schedule-dependent enhancement of antitumor activity of ethyldeshydroxy-sparsomycin in combination with classical antineoplastic agents.

The efficacy of the protein synthesis inhibitor ethyldeshydroxy-sparsomycin (EDSM) as a biochemical response modifier of several antitumor agents against L1210 leukemia and B16 melanoma is described. Seven drugs with different intracellular targets were selected for this combination study. Tumor implantation and drug treatment were both i.p., and the time interval between the administration of EDSM and the cytostatic agent was varied. Our results show that in the B16 tumor model EDSM is not able to potentiate any of these drugs, whereas antagonism is seen in combination with doxo-rubicin (DX). In the L1210 tumor model, however, no loss of activity is seen for this specific combination. The effect of the combination of cytosar (Ara-C), 5-fluorouracil (5-FU) or vincristine (VCR) with EDSM in the L1210 model is strongly time interval dependent. Loss of 5-FU antitumor activity is seen when EDSM is given 3 or 24 h after 5-FU; however, no effect is observed when EDSM is given 6 h after 5-FU. Enhancement of the 5-FU activity is not noticed. The VCR activity is potentiated when EDSM is given at least 6 h after VCR administration, which increases the antitumor response from 32 to > 60 days and the percentage survivors from 33 to 83% (p = 0.04). In combination with Ara-C, potentiation of antitumor activity is seen only when EDSM is given 24 h after Ara-C, which increases the antitumor response from 32 to > 55 days and the percentage survivors from 11 to 50% (p = 0.008). No modulatory effects are found when EDSM is combined with carmustine or DX.(ABSTRACT TRUNCATED AT 250 WORDS)

Preclinical antitumor activity of ethyldeshydroxysparsomycin in combination with cisplatin.

The efficacy of cisplatin (CDDP) in combination with the protein synthesis inhibitor ethyldeshydroxysparsomycin (EDSM) has been tested in two tumor models at various schedules. Mice with L1210 leukemia or B16 melanoma were treated with CDDP alone or in combination with EDSM. Against L1210 leukemia, which is sensitive to CDDP, combinations elicited increases in life-span for all treatment schedules compared to those achieved with the corresponding dose of CDDP. Moreover, the combination of EDSM with this platinum compound yielded a cure rate > 80%, compared to < 35% for single CDDP treatment. Although the B16 melanoma is rather resistant to both CDDP and EDSM, combinations of these agents against B16 melanoma showed schedule dependent efficacy and in certain schedules significant therapeutic advantage over individual drug treatment, but cures were not observed. Our results suggest that EDSM has significant synergistic capabilities in both animal tumor models, but strong therapeutic enhancement of cisplatin efficacy is only seen when the tumor is sensitive to CDDP.

Concentration and sequence dependent synergism of ethyldeshydroxy-sparsomycin in combination with antitumor agents.

The modulating effect of ethyldeshydroxy-sparsomycin (EDSM), an inhibitor of ribosomal protein synthesis, on cytostatic agents was studied on cultured B16 melanoma cells using the microculture tetrazolium test (MTT). The data were analyzed for true synergism using the combination index and the median effect principle. The extent of cytotoxic drug interaction was influenced by the duration of drug exposure, the dose ratio, as well as the treatment schedule. When drug ratios were used, synergism was observed upon pre- and post-treatment in combination with cisplatin, cytosine arabinoside (Ara-C), methotrexate (MTX) and 5-fluorouracil (5-FU). The combination of a fixed dose of EDSM was synergistic with cisplatin, Ara-C, vincristine (VCR) and MTX, in the order MTX > Ara-C > VCR > cisplatin, while the combinations with doxorubicin, 5-FU and etoposide (VP-16) were shown to be antagonistic. These results suggest that only certain drugs and treatment schedules might be worthwhile for combination studies with EDSM in vivo and indicate a role for EDSM as modifier of antitumor responses in cancer chemotherapy.

Correlation of the in vitro cytotoxicity of ethyldeshydroxysparsomycin and cisplatin with the in vivo antitumour activity in murine L1210 leukaemia and two resistant L1210 subclones.

The cultured murine leukaemia L1210 cell populations used in the present study were derived from L1210 cells that had been grown in vivo. Subclones resistant to sparsomycin (L1210/Sm) or cisplatin (L1210/CDDP) were also developed in vivo. The doubling times of the cultured cell populations were identical. Fractions surviving after drug treatment in vitro were determined by colony formation in soft agar. The results, based on the differential sensitivity of the cell populations to ethyldeshydroxysparsomycin (EdSm) and CDDP, indicated that after a short exposure, cultured L1210/CDDP cells were cross-resistant to EdSm. L1210/Sm cells, however, were not cross-resistant to CDDP. The results obtained in cultured cell populations were confirmed in vivo. CD2f1 mice bearing i.p. implants of 1 x 10(5) tumour cells were given EdSm or CDDP and a combination of the two agents. Drugs were given once daily every 4 days for 3 doses starting at 24 h after tumour implantation. Treatment of mice bearing L1210/wt leukaemia with combined EdSm and CDDP caused strongly synergistic antitumour activity. In animals bearing the two resistant subclones, however, combined drug treatment did not improve the antitumour activity. The corresponding median survival of mice receiving combined drug treatment was 60 days in each group containing 6 mice bearing L1210/wt, with 4-6 cures being noted; 19 days in animals harbouring L1210/Sm, with 2 cures being recorded among 6 mice; and 11 days in mice bearing L1210/CDDP, with no cure being obtained. The results of this study indicate that the synergism resulting from combined treatment with CDDP and EdSm is a function of the cellular properties of the target tumour-cell populations and is independent of host factors.

Potentiation of cisplatin antitumour activity by Ethyldeshydroxy-Sparsomycin in L1210 leukemia.

The combination of Ethyldeshydroxy-Sparsomycin (EdSm) with cisdiamminedichloroplatinum(II) (CDDP) caused significant antitumour activity against murine L1210 leukemia. Although single drug treatment by cisplatin generated some cures, all schedules of combined treatment, using nontoxic doses of EdSm (5mg/kg) and cisplatin (3 mg/kg), resulted in the cure of 4 to 6 mice in each group consisting of 6 mice. No differences in antitumour activity were observed between pretreatment, simultaneous treatment or posttreatment of cisplatin with EdSm. Increasing the number of tumour cells implanted i.p. diminished the antitumour effect of both EdSm as well as CDDP, but not for the drug combination. Changing the route of administration from i.p. to i.v. for one of the drugs out of the combination resulted in loss of antitumour activity.

In vitro studies on the influence of doxorubicin in combination with recombinant interferon-gamma on human monocytes.

The maturation and differentiation process of human monocytes and human monocyte-derived macrophage cytotoxicity mediated by receptors for the Fc moiety of immunoglobulin G (Fc gamma Rs) were studied in vitro using the chemotherapeutic drug doxorubicin in combination with the biological response modifier (BRM) recombinant interferon-gamma (rIFN-gamma). Human monocytes were cultured for 9 days and treated with doxorubicin before, after, or on day 7 of the culture. rIFN-gamma was added continuously on day 0 or on day 7 of the culture, either alone or in combination with doxorubicin. In the presence of doxorubicin, all measured intracellular enzyme activities were at the same level as the controls and the rIFN-gamma treated cells. However, when monocytes were co-cultured for 9 days with rIFN-gamma alone or in combination with doxorubicin, enzyme levels decreased to between 45% and 61% of the controls. In control cultures ADCC activities against maximally sensitized hRBC mediated by Fc gamma RI and Fc gamma RII were 41.7 +/- 8.8% and 42.7 +/- 11.6%, respectively. When monocytes were exposed to rIFN-gamma continuously or 40 h before harvesting, Fc gamma RI ADCC activity increased to 78.9 +/- 9.8% and 68.1 +/- 8.1%, respectively, and Fc gamma RII ADCC activity to 56.3 +/- 8.4% and 55.4 +/- 7.3%, respectively. The addition of doxorubicin to monocyte cultures in the presence or absence of rIFN-gamma did not influence the lysis of the two types of sensitized hRBC. These observations indicate that doxorubicin does not negatively influence the activation state of monocytes/macrophages, induced by rIFN-gamma.

Effects of doxorubicin on maturation of human monocytes in adherent and non-adherent cultures.

Purified human monocytes were cultured for 2 h, 88 h, and 10 days in plastic tubes (adherent) and for 10 days in Teflon foil bags (non-adherent). Monocytes were incubated with doxorubicin by two short-term exposures (750 or 1500 ng/ml) for 1 h or by continuous exposure (75 ng/ml). Maturation was monitored by measuring the intracellular activity of three metabolic enzymes and two acid hydrolases. Expression of receptors for the Fc moiety of immunoglobulin G (FcRI, FcRII, FcRIII), CD14, and HLA-DR was assayed by indirect immunofluorescence with monoclonal antibodies. In the presence of doxorubicin, the adherent capacity, the yield, and the enzyme activities reflecting growth and intermediary metabolism were similar to the control groups. However, doxorubicin reduced the expression of FcRI (32-45%), FcRII (10-26%), CD14 (20-37%), and HLA-DR (25-34%) on the monocyte-derived macrophages. Expression of FcRIII was not detectable after 10 days of culture.

Modulation of the in vitro cytotoxicity of seven anticancer drugs by protein synthesis inhibition using sparsomycin.

Inhibitors of protein synthesis may modify cell response to cytotoxic drugs. The influence of protein synthesis inhibition using sparsomycin (Sm) on the cytotoxicity of seven classical cytotoxic drugs, 5-FU, ARA-C, MTX, doxorubicin, melphalan, bleomycin and vincristine, was studied. Preincubations, simultaneous incubations and postincubations with Sm were investigated in vitro on CHO cells. Preincubation with Sm antagonized the activity of the S phase specific drugs 5-FU, ARA-C, MTX as well as vincristine, while postincubation with Sm enhanced their effect. A similar pattern was observed with doxorubicin. Preincubation with Sm had a potentiated non-S phase specific like bleomycin and cisplatin, but not melphalan. Postincubation with Sm had a potentiating effect on bleomycin but had no effect on melphalan. These results indicate a strong, schedule dependent effect of Sm on various drugs and suggest some potentially useful combinations to be tested in vivo.

Studies on retinotoxic potential of a novel antitumor antibiotic--sparsomycin--in rats.

Sparsomycin (Sm) is a potent inhibitor of protein synthesis with an anticancer potential. Two years after its discovery in 1962 a phase I clinical trial revealed serious drug-induced retinotoxicity. The mechanism of this toxicity still remains unresolved; however, its understanding is important for the reintroduction of Sm or one of its analogues in clinical practice. If Sm penetrates the retina, its toxic effect should be seen as inhibition of a protein(s) vital for the visual function. Treatment of healthy albino rats and Royal College of Surgeon (RCS) rats with subtoxic doses of Sm was unable to produce ocular toxic effects. Disruption of the blood-retina barrier with sodium iodate allowed Sm to decrease opsin content by only 27%. These results strongly indicate that Sm might become retinotoxic solely upon extreme conditions such as permeabilization of the blood-retina barrier which may happen only in some rare pathological situations.

In vivo antitumor activity of sparsomycin and its analogues in eight murine tumor models.

Sparsomycin (Sm) is a known inhibitor of ribosomal protein synthesis with an attractive anticancer potential. Recently, several analogues of Sm which are more active than the parent drug were selected for further study on the basis of in vitro investigations. Six analogues as well as the parent drug were tested for their antitumor activity in eight in vivo murine tumor models: P388 and L1210 leukemias, RC renal cell carcinoma, B16 melanoma, C38 colon carcinoma, LL Lewis lung carcinoma, C22LR osteosarcoma and M5076 sarcoma. Sm itself appeared to have only borderline activity on L1210 leukemia. The analogues that were most active in vitro showed also the highest in vivo activity. The most sensitive tumors were RC, L1210 and P388. Minimal activity was found on B16 and no activity on C22LR, M5076, C38 and LL. The most active compounds are deshydroxy-Sm, ethyl-deshydroxy-Sm and n-pentyl-Sm. There was a considerable loss of activity when L1210 leukemia was implanted sc while the drugs were administered iv. Only one drug, ethyl-deshydroxy-Sm appeared to be active in this assay. No single most effective compound could be found in this study. The overall activity of Sm and its analogues is moderate. The three analogues which show high activity in three ascitic tumors will be further investigated using human tumor xenograft models.

Modulation of placental alkaline phosphatase activity and cytokeratins in human HN-1 cells by butyrate, retinoic acid, catecholamines and histamine.

The effects of butyrate and retinoic acid in combination with catecholamines or histamine on the HN-1 human head and neck squamous carcinoma cell line were investigated analysing cell proliferation, placental alkaline phosphatase (PLAP) activity, and relative cytokeratin content. Butyrate inhibited cell proliferation in agar, whereas retinoic acid induced a small inhibitory effect. Butyrate enhanced PLAP activity in a time related manner in contrast to retinoic acid, which had no significant effect. However, retinoic acid inhibited the efficacy of butyrate to induce PLAP activity. A synergistic enhancement of PLAP activity was demonstrated after treatment of butyrate pretreated cells with catecholamines or histamine. The beta-adrenergic antagonist propranolol partly inhibited the aforementioned enhancement of PLAP activity, whereas the alpha-adrenergic antagonist phentolamine further enhanced PLAP activity. Indirect labeling of keratins with a polyclonal antibody showed that cytokeratin content was enhanced by butyrate but not by retinoic acid. Further analysis of cytokeratin content using four monoclonal antibodies showed that labeling of cytokeratins (5 + 8) was increased by butyrate. PLAP activity could be modulated by a concerted action of either butyrate plus retinoic acid or butyrate plus catecholamines or histamine, indicating a possible role for PLAP in tumour cell proliferation.

In vitro modulation of cisplatin cytotoxicity by sparsomycin inhibition of protein synthesis.

Inhibition of protein synthesis can alter cellular responsiveness to the classical anticancer drugs. The in vitro response of Chinese hamster ovary (CHO) cells to cisplatin with or without sparsomycin (Sm) was studied with the use of [3H]leucine and [methyl-3H]thymidine incorporation and clonogenic assay. Pretreatment of exponentially growing CHO cells with 1 microgram Sm/ml for 3 or 5 hours decreased [3H]leucine incorporation by 20% and resulted in significant resistance to cisplatin (P = .005). Sm in a concentration of 10 micrograms/ml reduced [3H]leucine and [methyl-3H]thymidine incorporation after 3 hours by 92 and 84%, respectively, and resulted in potentiation of the cisplatin cytotoxicity (P = .004). This effect was the same in the case of nonproliferating cells (P = .005), while protection due to Sm (1 microgram/ml) was seen only during cell proliferation. Simultaneous incubation and postincubation with Sm proved to have much less or no potentiating effect on cisplatin. The mechanisms of both protection and potentiation are still not clear, but our data indicate that Sm is a promising drug for further studies on the modulation of the cancer cell response to classical anticancer drugs.

Pharmacokinetics and toxicology of sparsomycin in beagle dogs.

Sparsomycin is a cytotoxic drug exhibiting a broad spectrum of in vitro activity against murine tumors and many tumor cell lines. It also appears to be a potent stimulator of the antitumor activity of cisplatin against L1210 leukemia in vivo. However, because of its toxicity, the antitumor activity of sparsomycin on murine tumors in vivo has been disappointing. The purpose of our study was to investigate the pharmacokinetics of this drug as well as the possible mechanisms that produce sparsomycin toxicity. Tests on beagle dogs revealed that about 60% of the drug is eliminated by metabolic clearance, while 40% is eliminated by the kidneys. After a single bolus injection of 0.1 mg/kg sparsomycin without narcosis, sparsomycin was eliminated with a t beta 1/2 of 0.6-0.7 h, the AUC being 0.32-0.38 mg.h.l-1, and the volume of distribution (Vd) 0.26 l/kg. In addition to being subject to glomerular filtration, sparsomycin is probably also actively excreted and actively reabsorbed by the renal tubuli. Sparsomycin itself may inhibit its active tubular excretion, thus resulting in a decrease in the drug's renal clearance and its accumulation in the plasma. Sparsomycin appeared to be toxic primarily in the liver, disturbing its function and the synthesis of plasma proteins. Two out of five dogs developed hemorrhagic diathesis due to hypofibrinogenemia and deficiency of other blood-coagulation factors. Sparsomycin was not toxic to the bone marrow.

In vivo potentiation of cis-diamminedichloroplatinum (II) antitumor activity by pretreatment with sparsomycin.

The influence of protein synthesis inhibition by sparsomycin (Sm) on in vivo cisplatin activity has been studied on BALBc X DBA2: F1 mice bearing L1210 leukemia i.p. Sm alone at the dose range from 0.5 to 3.0 mg/kg did not significantly improve animal survival. Sm potentiated cisplatin activity only when given 3 or 6 h prior to cisplatin (P less than 0.001). Sm 0.5-1.5 mg/kg 3 h prior to cisplatin resulted in a significant prolongation of animal survival (P less than 0.001) and 66% cures in each group versus 0% due to cisplatin alone. Sm pretreatment decreased weight loss due to cisplatin suggesting that it probably is able to decrease cisplatin toxicity.

Flow cytometric evaluation of cell dispersion from human head and neck tumors.

The preparation of single-cell suspensions from 25 human head and neck tumors is described. Dispersal was performed overnight at 4 degrees C under slight agitation of the tissue suspensions using various combinations of enzymes and additives. The cell suspensions were examined for number of cells released, viability, amount of debris, and DNA distribution by means of flow cytometry (FCM). It was shown that both trypsin/dithioerythritol (TD) and collagenase/D Nase (CDse) were of value in dispersing single cells from tumor tissue. In contrast to CDse, incubation with TD appeared to be cytolytic to normal lymphocytes. In a number of cases, DNA-FCM revealed ploidy abnormalities in a TD-suspension, which were not discernible in the concurrent CDse-suspension. Cell culture of primary cell suspensions corroborated the reliability of the DNA-FCM measurements. Pretreatment with CDse improved tumor disaggregation by TD and indicated a different dispersal capacity. Addition of Ca2+ and Mg2+ ions to the dispersal mixtures and preincubation of tumor slices in complete medium for 1 day before initiation of cell dispersion influenced favorably the quality of the cell suspension.

Metabolic changes in the psoriatic lesion during therapy.

Three marker enzymes were measured during treatment of psoriatic plaques with two different therapies. During treatment with clobetasol propionate the epidermal enzymes (acid phosphatase and glucose-6-phosphate dehydrogenase) returned to normal within 14 days whereas capillary alkaline phosphatase remained at the original level. By contrast, all three marker enzymes reverted to normal at the same tempo during PUVA therapy, reaching the control range after 4-8 weeks.

Monocyte function is normal in quiescent psoriasis.

We report an investigation of peripheral blood monocytes from untreated patients with mild, quiescent psoriasis. Possible metabolic changes were monitored by the determination of 3 enzymes representing different pathways of glucose metabolism and 2 lysosomal enzymes. Signal processing was evaluated by the measurement of cyclic AMP levels before and after hormonal stimulation. Luminol-amplified chemiluminescence provided an objective approach to assessing phagocytic capacity. Finally, the pattern of maturation of normal and psoriatic monocytes has been compared during culture in vitro. Our results were uniformly and wholly negative; we conclude that the concept of an "intrinsic" abnormality of the psoriatic monocyte may be excluded. Possible reasons for discrepancies in the literature are discussed.

Characterization of monocyte maturation in adherent and suspension cultures and its application to study monocyte differentiation in Hodgkin's disease.

Monocytes purified with cell scatter monitored counterflow centrifugation were cultured in plastic (adherent) and in teflon culture bags (suspension). Sequential changes were monitored during 15 days by measuring intracellular activity of three enzymes of intermediary metabolism: glucose-6-phosphate dehydrogenase (G-6-PDH), phosphohexose isomerase (PHI) and isocitrate dehydrogenase (ICDH), and the two acid hydrolases: acid phosphatase (ACP) and N-acetyl-beta-glucosaminidase (NAG). In teflon grown macrophages a significantly lower G-6-PDH activity was seen after 15 days in comparison to plastic adherent macrophages (P less than 0.0002). For the other enzymes similar values for both culture modalities were found. The significantly, cycloheximide insensitive, higher values for G-6-PDH, PHI and ICDH in 2 h plastic adherent monocytes in comparison with plastic non-adherent monocytes, suggest a relationship between adherent capacity and the level of intermediary metabolism. The overall yield of plastic adherent macrophages after 15 days was 35% in contrast with 89% for the in suspension cultured macrophages. This corroborates the existence of adherent and non-adherent monocytes, both capable of differentiation in vitro. In 14 patients with advanced Hodgkin's disease (HD) and 14 normal controls, monocyte differentiation was studied applying both culture modalities. The enzyme levels, reflecting growth and intermediary metabolism, were similar for both groups. The adherent capacity and yield, both in teflon and in plastic, after 15 days was comparable for both groups. It was concluded that in vitro monocyte differentiation in the presence of autologous serum was qualitatively and quantitatively normal in advanced HD; this is in favour of an intrinsically normal function of monocytes in HD.

Response of the clinically uninvolved skin of psoriatic patients to standardized injury.

Test sites on healthy controls and on the clinically uninvolved skin of psoriatic patients were stripped with tape, and eight variables were quantified at intervals during the subsequent healing process. In the control groups, the stratum corneum regenerated at a constant rate and the underlying skin showed elevations of metabolic activity peaking around days 2-4. In the psoriatic groups, we observed that (I) the response of the keratinizing zone is identical to that of the controls, (2) the proliferative response is initially normal but remains elevated rather longer than usual, and (3) the dermal capillaries (indicated by alkaline phosphatase activity) show a gross hyper-reactivity which is already apparent after 1 day and which persists for more than a week. These findings support our previous conclusion that metabolic alteration of the dermal capillary precedes epidermal hyperplasia in the pathogenesis of the psoriatic lesion.