John S. Lazo

Endothelial injury caused by antineoplastic agents

The vascular lining of the blood vessels to normal organs and malignant tissues would be expected to reflect the functional demands placed upon it. These functional requirements may be accomplished by specific biochemical macromolecules, some of which are localized on the plasma membrane of the endothelium. Considerable evidence exists that toxicity to at least some normal organs caused by antineoplastic agents is heralded by endothelial damage. This endothelial damage may reflect a specific drug-endothelium interaction, the mechanism and basis of which are not yet understood. The possibility also exists that destruction of solid tumors by currently employed antitumor agents is mediated in part by local loss of essential vasculature. Selective destruction of the tumor endothelium could be a rational and novel target to which drugs could be designed.

Alterations in pulmonary protective enzymes following systemic bleomycin treatment in mice

Repeated bleomycin administration in animals and humans produces significant lung fibrosis. The pathogenesis of this toxicity may be multifactorial, but it appears to be initiated through the production of radical oxygen species by an activated bleomycin-iron-oxygen ternary complex. Protection of lung tissue from bleomycin-induced toxicity may occur through both specific metabolic inactivation of bleomycin by the enzyme bleomycin hydrolase, as well as by such non-specific antioxidants as catalase and the glutathione system. The effect of chronic, systemic administration of bleomycin on the activities and levels of these enzymes and proteins in pulmonary tissue is unknown. C57BL/6 mice were injected subcutaneously with saline, non-fibrogenic (2 mg/kg) and fibrogenic (10 mg/kg) doses of bleomycin twice-weekly for 6 weeks. Animals were killed at 0, 1.5, 3, and 6 weeks after initiation of bleomycin treatment. Catalase activity was increased more than 50% at 3 weeks in the low-dose animals, and was decreased over 40% at 6 weeks in the high-dose animals. Total lung glutathione levels were unaffected in both groups, although glutathione reductase activity was increased significantly (over 2-fold) at 1.5 and 3 weeks in the high-dose animals. At 6 weeks glutathione reductase was increased 7- and 12-fold in low and high-dose animals respectively. Glutathione peroxidase activity also was elevated more than 2-fold above control values at 6 weeks in both sets of animals. There was no evidence of induction of bleomycin hydrolase activity at any time point. Rather, bleomycin hydrolase activity was decreased significantly to 30 and 40% of control values at 3 and 6 weeks, respectively, in mice receiving the fibrogenic doses of bleomycin. These results demonstrate that chronic, systemic administration of non-fibrogenic and fibrogenic doses of bleomycin produces changes in activity of lung antioxidant defense mechanisms. The early loss of lung bleomycin hydrolase activity may contribute to the pathogenesis of bleomycin-induced pulmonary toxicity following repeated drug exposure.

Reduction in rabbit serum and pulmonary angiotensin converting enzyme activity after subacute bleomycin treatment

Abstract We studied morphological and biochemical alterations in the lungs of rabbits treated subcutaneously with bleomycin (5 mg/kg) three times weekly for 28 days. This treatment produced morphologically evident pulmonary damage characterized by the appearance of ciliated alveolar epithelial cells and endothelial blebs. No change in pulmonary collagen content was evident. Rabbits treated with bleomycin had significantly less pulmonary angiotensin converting enzyme activity than control animals, although no alterations in lung DNA, protein, glycosaminoglycans or hydroxyproline (an index of collagen) were observed. Serum angiotensin converting enzyme activity decreased with repeated bleomycin administration and was reduced by 42, 48 and 65 per cent from control animals 2, 3 and 4 weeks, respectively, after the initiation of drug treatment. These data provide biochemical evidence that subacute bleomycin administration damages the pulmonary endothelium in the absence of fibrosis and can reduce the serum activity of angiotensin converting enzyme in rabbits.

Phase I trial of combined therapy with bleomycin and the calmodulin antagonist, trifluoperazine

SummaryCalmodulin antagonists, such as trifluoperazine, can enhance the cytotoxic effects of bleomycin both in tissue culture and in vivo. Therefore, we evaluated the effects of combination treatment with these drugs in a phase I clinical trial. Patients with objectively measurable or evaluable cancer refractory to conventional treatment who had an acceptable performance status (ECOG 0-2) and acceptable laboratory studies were eligible. All patients gave written informed consent. A cycle of therapy consisted of three weekly treatments with trifluoperazine (days 1–4) and 30 IU bleomycin (day 3). After three patients completed a cycle of therapy without experiencing dose-limiting toxicity, new patients were entered in the study and received a higher dose of trifluoperazine. The dose of bleomycin remained constant. Evaluable patients received at least 2 weeks of treatment and survived for 6 weeks; of 19 patients, 2 were unevaluable. The major toxicities were neurological and pulmonary and included one case of fatal pneumonia with interstitial pulmonary fibrosis. There was no hematologic toxicity. Two patients underwent partial responses (PRs) and two had complete responses (CRs). We conclude that trifluoperazine can safely by given with bleomycin and that further study of the potential efficacy of this treatment is indicated.

Angiotensin converting enzyme activity in mice after subacute bleomycin administration

Abstract Multiple ip or sc injections of bleomycin (BLM) in mice elevated the serum and pulmonary activities of the peptidyl dipeptidase, angiotensin converting enzyme (ACE). ACE activity in lungs of mice treated every 3 days with BLM (9 mg/kg) was elevated 33, 36, and 45 days after the initiation of ip drug administration. After 12 BLM injections the increases in enzyme activities were dose related, with mice receiving the largest cumulative dose (108 mg/kg) having the greatest increase (53%) in lung ACE activity above control values. Mice treated every 3 days sc also had elevated pulmonary ACE activities after 15 injections, although liver and spleen activities were unaltered and the activities in the kidneys were reduced over 70% from control values. In addition to elevated lung ACE activity, serum ACE activity was increased 22 and 26% in mice treated ip and sc, respectively. A marked elevation in lung collagen as measured by lung hydroxyproline content was detected at the time of increased lung and serum ACE activity. Mice injected ip more frequently but with a lower dose of BLM (1.5 mg/kg, 2×/day, 5 days/week × 5 weeks) displayed no change in pulmonary ACE activity and hydroxyproline content even though they had received, over the same time period, a cumulative dose of BLM equal to that achieved by injecting 9 mg/kg of BLM every 3 days × 12. These data indicate that, subacute treatment of mice with BLM leads to an elevation in pulmonary and serum ACE activity and a decrease in renal ACE activity at the time of the development of fibrosis. The increase in lung ACE activity and hydroxyproline content appears to depend not only upon the total cumulative dose but also the dosage schedule. The elevation in serum ACE activity may reflect the loss of kidney enzyme or the shedding of pulmonary enzyme.

Aromatase activity in human ovarian cancer

Eighty-four tumor samples from 70 women with primary ovarian cancer were assayed for cytosol estrogen (ERc) and progestin (PRc) receptor concentrations and aromatase activity. In addition, 22 of the tumors were studied for their response to the aromatase inhibitor, 4-OH-androstenedione, in a soft agar clonogenic cell assay system. Although aromatase activity was detected in almost all of the primary tumors, this enzyme was barely detectable in the majority of metastatic tumor samples. There was no significant correlation between aromatase activity and either the ERc or PRc content of the tumors, or tumor grade. Of 12 tumors grown successfully in the soft agar culture system, only 1 showed a substantial (greater than 50%) reduction in colony-forming efficiency after exposure to the aromatase inhibitor. These results suggest that local estrogen biosynthesis probably does not play an important role in the majority of epithelial ovarian tumors. However, there may be a small subset of estrogen receptor-positive tumors in which aromatase could provide a local growth stimulus.

Chemical modulation of bleomycin induced toxicity

Both lidocaine (LIDO) and the calmodulin (CaM) antagonists, pimozide (PIM) and trifluoperazine (TFP), enhanced bleomycin (BLM) induced cytotoxicity and DNA damage. The toxicity with the combination of BLM and CaM antagonists represented true pharmacological synergism and was observed with the addition of the CaM antagonist either during or after BLM exposure. Additionally, the DNA damage of BLM and the BLM-like drugs, talisomycin S10b (TAL) or peplomycin (PEPLO), was also enhanced by CaM antagonists. LIDO, which similarly increased the lethal effects and DNA damage of BLM in L1210 cells, was also effective only during or after BLM exposure. The data presented here indicate that the modulation of toxicity seen with these drug combinations is reflected by changes in DNA integrity. Furthermore, these data suggest that the inhibition of DNA repair processes may be at least partially responsible for the enhanced toxicity and DNA damage when CaM antagonists or LIDO are added to BLM.

Solubilization of pulmonary angiotensin-converting enzyme with 1-O-n-octyl-β-d-glucopyranoside

Abstract Ninety percent of rat pulmonary angiotensin-converting enzyme was solubilized in a single step using the nonionic detergent 1- O - n -octyl-β- d -glucopyranoside. This detergent has low absorbance at 228 nm and, thus, is compatible with the commonly used spectrophotometric assay of D. W. Cushman and H. S. Cheung (1971, Biochem. Pharmacol. 20 , 1637–1648). The maximum solubilization occurred with 30 m m 1- O - n -octyl-β- d -glucopyranoside and at this concentration of detergent a four-fold increase in specific activity was observed.

Effect of Fluosol-DA/O2 on the antitumor activity and pulmonary toxicity of bleomycin

SummaryThe effect of an oxygen-carrying perfluorochemical emulsion on bleomycin antitumor activity and pulmonary toxicity was examined. Fluosol-DA (0.3 ml/mouse, i. v.), combined with bleomycin (10 or 15 mg/kg, i. p.) and a 2 h exposure to 95% oxygen (BFO) increased by five- to six-fold the tumor growth delay of FSaIIC fibrosarcoma compared to bleomycin alone (B). Only a slight increase in tumor growth delay was noted with the incomplete combinations of bleomycin and O2 (BO) and bleomycin and Fluosol-DA (BF). When bleomycin (10 mg/kg) was co-administered with 0.3 ml Fluosol-DA and 95% O2, cell survival was reduced ten-fold compared to that seen with bleomycin alone. In contrast, the surviving fraction of cells obtained from FSaIIC tumors treated in vivo indicated that 0.3 ml Fluosol-DA per mouse or a 2 h exposure to 95% O2 did not markedly alter the effects of bleomycin alone.The pulmonary effects of the BFO combination were assessed during the course of the therapy by bronchoalveolar lavage (BAL) analysis and pulmonary hydroxyproline (OH-Pro) content. Mice treated with this combination had a 20-fold increase in total numbers of cells obtained in the BAL compared to control animals. An increased cellularity in the lungs was also seen morphologically. The composition of the cells in the lavage fluid was altered after BFO but not BO treatment and reflected a neutrophilic influx. Furthermore, total protein recovered in the BAL fluid was increased 5-fold in the BFO treatment group compared to that in the control mice. Pulmonary OH-Pro, an index of collagen and fibrosis, was unaffected acutely after three treatments of either BFO or BO compared to control mice. Thus, Fluosol-DA and O2 can enhance the antitumor activity of bleomycin. The increased pulmonary cellularity suggests that this combination may also have adverse effects on lung tissue.

Reduction in cell surface Concanavalin A binding and mannose incorporation into glycoproteins of Sarcoma 180 by 6-thioguanine

Abstract Treatment of mice bearing Sarcoma 180 ascites cells with 20 mg kg of 6-thioguanine for 1 hr caused a 50 per cent decrease in [2-3H]mannose incorporation into glycoproteins; this inhibition continued for at least 6 hr after drug exposure. The decrease in labeling of mannose-containing glycoproteins produced by the 6-thiopurine was followed by a decrease in the specific binding of Concanavalin A to Sarcoma 180; this effect was observed as early as 12 hr after 6-thioguanine treatment. The decrease in specific binding of Concanavalin A to Sarcoma 180 24 hr after 6-thioguanine was due to a 20 per cent reduction in the number of Concanavalin A receptors available on the plasma membrane of these neoplastic cells. The binding of lectins specific for sugars not activated by GTP, such as Wheat Germ Agglutinin and Ricinus communis, was not decreased by treatment of cells with the purine antimetabolite. This disruption in the biochemical composition of the cell surface produced by 6-thioguanine was accompanied by morphological alterations in the microvillar content of Sarcoma 180 cells which were observed by scanning electron microscopy. The alterations in membrane composition and structure caused by this antineoplastic agent are hypothesized to be of importance in the delayed cytotoxicity of 6-thioguanine.