Radiol Oncol 1993; 27: 271-4. Changes in the quantity of cathepsin D in irradiated human cells following treatment with hyperthermia and interferon a Ana Ferle-Vidovič,1 Marija Kaštelan,1 Danilo Petrovič,1 Janez Škrk,2 Ivan Vrhovec2 1 Ruder Boškovic Institute, Zagreb Croatia, 2 Institute of Oncology, Ljubljana, Slovenia In the present work, the changes in quantity of cathepsin D, an aspartic proteinase, in proliferating human nonmalignant (HEF) and malignant (HEp2) cells after combined treatment by gamma irradiation, hyperthermia and by interferon a-2b (IFNa) were followed. Correlation between the antiproliferative effect of these combined agents and changes in the concentrations of this cathepsin D were expected. Evidently, the treatment of cells in culture by IFNa, combined with irradiation and elevated temperature, produces an increased quantity of cathepsin D. In nonmalignant HEF cells these effects are more expressed than in malignant HEp2 cells. Key words: cathepsin D; cell, cultured-radiation effects; hyperthermia, induced; interferon-alfa-2B Introduction Intracelular proteinases participate in the vital cellular processes such as growth and multiplication, response to DNA damage and radiation -response. '' 2' 3' 4 Recent studies suggest that the aspartic proteinase cathepsin D, may also be implicated in the process of tumor invasion and metastasis, 5 Several in vitro observations showed that this proteinase may facilitate the spread of neoplastic cells through different rnec-hanisms releated to its proteolytic activity, by acting at different levels of the metastatic cascade. Cathepsin D was also shown to be able to degrade in vitro the extracellular matrix, and Correspondence to: Ana Ferle Vidovic, Ph. D., Ruder Boskovic Institute, Bijenicka cesta 54, 41000 Zagreb, Croatia UDC 615.281.7:577.156.2 to activate latent precursor forms of other pro-teinases involved in the invasive steps of the metastatic process. Interferon a-2b (IFNa) initially recognized for its antiviral effects, has also been shown to have antiproliferative, imrnunoregulatory and antitumor activities.6 There is some experimental evidence supporting the concept that modest levels of hyperthermia might be beneficial to the action of interferons. This concept is supported by in vivo experiments showing that modest levels of hyperthermia enhanced the action of interferon.7 In vitro it was found that hyperthermia acted synergistically by enhancing the proliferative effects of IFNa.8 There is definitive experimental evidence in vivo for synergistic effect of this combined treatment.9 Similar synergistic effects were also observed, when interferon was applied in combination with irradiation. 10 272 Ferle-Vidovic A. el al. Such results may have clinical importance, because they suggest that hyperthermia could be used in combination with IFNa. to provide a synergistically enhanced antitumor action.9' 10 Therefore, it is possible that the combination of hyperthermia and IFNa therapy may have clinical application in cases when technically feasilbe. Such combined treatment can, however, induce an increase in proteolitic enzymes in cells, which might enhance metastasing of the treated tumor. In this work we determined the changes in the quantity of the aspartic intracellular proteinase - cathepsin D in irradiated human non-malignant (HEF) and in malignant (HEp2) cells after combined treatment with hyperther-mia and/or IFNa.. Correlation between the antiproliferative effect of these combined agents and the changes in concentrations of this cat-hepsin was found. Materials and methods Cell cultures and experimental procedure Human embrional fibroblasts (HEF) and human laryngeal carcinoma (HEp2) cells, were cultured as monolayers in Eagle's minimal essential medium, supplemented with 10 %% foetal calf serum. Celi cultures were prepared by plating 105 of 106 cells per Petri dish of 10 cm in diameter (3 dishes per experimental point) and after two days of growth, cells were irradiated, treated by IFNa. and by hyperthermia in the following combinations: irradiation only, IFNa. only, irradiation plus IFNa., irradiation plus IFNa. plus hyperthermia. Following the mentioned treatment, celi cultures (106 per dish) were kept at 37° C and samples taken after one hour were stored at -20° C until proteinase quantity assay. The number of proliferating cells (105 per dish) following irradiation and combined treatment were counted 24 and 96 hours after treatmen. Interferon Recombinant interferon a.-2B (INTRON-A, Schering-Plough-Baltimore-USA) was added to the growth medium to reach final concentration of 1 X 104IU/ml. Cells were incubated in the IFNa.-containing medium at 37° C for 1 hour and then incubated until additional treatment or harvesting. Hyperthermia Heat treatment was conducted by submerging the Petri dishes in a water bath at 44° C for 20 min. HEF and HEp2 cells were exposed simul-taneoulsy to IFNa. and to hyperthermia. Irradiation For gamma irradiation, a Gamma Celi 220 (Atomic Energy of Canada, a.td) unit was used. The dose rate was 4,13 Gy/min, with the total dose 15 Gy/sample for cathepsin D, or 5 Gy for the growth inhibing effect. Cathepsin D concentration Following treatment, celi cultures were incubated in the growth medium at 37° C. Samples were taken after different time intervals, placed on ice and washed three times with cold phosphate buffered saline. The cells were harvested by a rubber policeman, concentrated by centri-fugation (10 min at 1000 rpm), lysed in distilled water and frozen at -20° C until assay. The concentrations of cathepsin D were determined using specific enzyme immunometric assay (ELSA-CATH-D kit, CIS Bio International, Solid phase two-site immunoradiometric assay), for the quantitative determination of total cathepsin D in cytosol. Results The combined effects of IFNa, hyperthermia and irradiation on celi proliferation, expressed as the number of growing HEF celi population, are shown in Table 1. The antiproliferative effects were expressed as percentage of cell numbers in control samples. While irradiation or IFNa alone, moderately inhibited celi growth, the treatments, by IFNa plus irradia- Irradiation, hyperthermia, interferon a and cathepsin D 273 tion, and in particular, when lFNa was combined with irradiation and hyperthermia, the antiproliferative effect was markedly enhanced. Table 1. Cell numbers (x10 HEFcells) after combined treatment with irradiation and/or interferon a and/or hyperthermia. Group of Incubation time Percent of treatment after treatment control 24h 96h 24h 96h Controls 10,9 ± 2,6 20,4±3,1 - - Irradiation .5,0 ± 0,9 7,5 ±1,1 46 37 Interferon« 6,6 ± 0,2 8,7± 1,7 61 43 Irradiaton + Interferon« 3,7 ±0,1 4,0±0,4 34 20 Irradiation + Interferon« + 1,8±0,1 1,9±0,2 16 9 Hyperthermia___ The combined effects of lFNa, hyperthermia and irradiation on cell proliferation, expressed as the numbers of growing HEp2 cell population, are shown in Table 2. The antiproliferative effects were expressed as percentage of cell numbers in control samples. While irradiation or lFNa alone, moderately inhibited HEp2 cell growth, the treatmens by lFNa plus irradiation, and in particular when lFNa was combined with irradiation and hyperthermia, the antiproliferative effect was markedly enhanced. Changes in the concentrations of cathepsin D measured in irradiated, proliferating human nonmalignant (HEF) and human malignant (HEp2) cell lines following combined treatment by interferon a and by hyperthermia are shown in Table 3. Table 2. Cell numbers (xlO5 HEp2 cells) after combined treatment with irradiation and/or interferon a and/ or hyperthermia. Group of Incubation time Percent of treatment after treatment control 24h 96h 24h 96h Controls 16,4± 1,4 41,0± 4,2 _ Irradiation 12,0±0,9 18,6 ± 1,6 73 45 Interferon « 11,0±2,l 21,8 ±4,0 67 53 Irradiation + Interferon « 8,0±1,1 10,0 ±0,7 48 24 Irradiation + Interferon a + 3,6 ± 0,8 7,7 ± 0,7 22 18 Hyperthermia The changes in the concentrations of cathepsin D were dependent on the agent used. Gamma iradiation alone revealed little change in the concentration of cathepsin D in the malignant celi line (1.1), whereas the same changes were more evident in the nonmalignant cells (1.3). Interferon a increased the levels of cathepsin D in both cell strains. These effects are more expressed in malignant cells (1.5), than in nonmalignant cells (1.2). Interferon a plus irradiation increased the concentration of cathepsin D significantly more than in the former two cases, when the agents were applied separately. The increased concentrations of the enzyme were similar in malignant (1.6) as in nonmalignant (1.8) cells. Combined application of all three agents together was most effective in increasing the amounts of cathepsin tested. Most evident effects were achieved in both cell lines after combined treatment with all three agents and again, with more expressed effects Table 3. Changes in the quantity of Cathepsin D in irradiated HEF and HEp2 cells following combined treatment. Group of HEFcells HEp2 cells treatment Cathepsin D ng/mg proteins T/C* Cathepsin D ng/mg proteins T/C* Controls 105 ± 11 _ 562 ± 14 _ Irradiation 138 ±7 1,3 604 ± 12 1,1 Interferon a 127±9 1,2 875 ±22 1,5 Irradiation + Interferon a 198 ±26 1,8 856 ± 31 1,6 Irradiation + Interferon a + 332 ± 16 3,1 1407 ± 53 2,5 Hyperthermia * T/C = Treated/Control 274 Ferle-Vidovic A. et at. on the nonmalignant HEF celi line (3.1), the in the malignant HEp2 cells (2.5). Discussion Cathepsin D, as mentioned before, plays an important role in tumor invasion and metastasis. Conelation between elevated levels of the enzyme in tumor cells and their ability to meta-stasise were found.5 On the other hand in our previous experiments we found that agents used in tumor therapy can influence the concentrations of various intracellular proteolytic enzymes, either by increasing or decreasing their concentrations11 or activities.12 This raises the question, particularly when cathepsin D is concerned, whether a particular tumor treatment could perhaps, apart of its cell killing potential, have some unwanted effects due to possibly elevated levels of cathepsin D. Our results show that such agents (irradiation, interferon a and heath), when given in amounts that evidently produce celi growth inhibition, can significantly increase the intra-cellular concentrations of cathepsin D, and therefore could consequently enhance the potential of the tumor cells to infiltrate the neighbouring tissues or to metastasise. If this occurs in a tumor bearing organism, this should also be kept in mind, when predicting the outcome of a particular tumor therapy. This may be even more important at combined modality therapy regimens. Acknowledgement We thank Mrs. Ljiljana Krajcar for her excellent technical assistance. This project was supported by the Ministry of Science of the Republic of Croatia and the Ministry of Science and Technology of Republic of Slovenia. References 1. Holzer H, Heinrich PC. Control of proteolysis. Ann Rev Biochem 1980; 40: 63-91. 2. Korbelik M, Skrk J, Suhar A, Turk V. The role of proteinases, interferons and hormones in proliferative activities of nonmalignant and malignant cells. Neoplasma 1988; 35: 555-63. 3. Scher W, Scher BM, Waxman S. Proteases stimulate mouse erythroleukemia cell differentiation and multiplication. Biochem Biophys Res Comm 1982; 109: 348-54. 4. Walker CG. Induclible DNA repair systems. Ann Rev Biochem 1985; 54: 425-57. 5. 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