Ferle-Vidović, Ana

Kaštelan, Marija

Petrović, Danilo

Vrhovec, Ivan

Škrk, Janez

English

Digital repository of Slovenian research organizations

Radio/ Oncol 1993; 27: 271-4. Changes in the quantity of cathepsin D in irradiated human cells f ollowing treatment with hyperthermia and interferon a Ana Ferle-Vidovic, 1 Marija Kaštelan, 1 Danilo Petro vic, 1Janez Škrk,2 Ivan Vrhovec2 1 Ruder Boškovic Institute, Zagreb Croatia, 2 Institute of Oncology, Ljubljana, Slovenia In the presen! 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 antiproJiferative 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 a,;,d 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 vita! cellular processes such as growth and rnultipli­cation, response to DNA darnage and radiation -response. 1• 2• 3• 4 Recent studies suggest that theaspartic proteinase cathepsin D; rnay also beimplicated in the process of tumor invasion andrnetastasis, 5 Severa! in vitro observations s110-wed that this proteinase rnay facilitate thespread of neoplastic cells through different rnec­hanisms releated to its proteolytic activity, byacting at different levels of the rnetastatic casca­de. Cathepsin D was also shown to be able todegrade in vitro the extracellular rnatrix, andCorrespondence to: Ana Ferle Vidovič, Ph. D., Ruder Boškovic Institute, Bijenička cesta 54, 41000 Zagreb, Croatia UDC 615.281.7:577.156.2 to activate Iatent precursor forrns of other pro­teinases involved in the invasive steps of the rnetastatic process. Interferon a-2b (IFNa) initially recognized for its antiviral effects, has also been shown to have antiproliferative, imrnunoregulatory and antiturnor activities.6 There is some experirnen­tal evidence supporting the concept that rnodest levels of hypertherrnia rnight be beneficial to the action of interferons. This concept is sup­ported by in vivo experirnents showing that rnodest levels of hypertherrnia 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 experirnental evidence in vivo for synergistic effect of this cornbined treatrnent.9 Sirnilar synergistic effects were also observed, when interferon was applied in combination with irradiation. 10 272 Ferle-Vidovic A. et 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 thc treated tumor. In this work we determined the changes in the quantity of the aspartic intracellular pro­teinase - cathepsin D in irradiated human non­malignant (HEF) and in malignant (HEp2) cells after combined treatment with hyperther­mia and/or IFNa.. Correlation between the an­tiproliferative effect of these combined agents and the changes in concentrations of this cat­hepsin 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 hyperthennia. 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. Thenumber of proliferating cells (105 per dish)following irradiation and combined treatmentwere 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 fina! concentration of l 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 simul­taneoulsy to IFNa. and to hyperthermia. Jrradiation 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 medi um at 37° C. Samples were taken after different tirne intervals, placed on ice and washed three times with cold phos­phate buffered saline. The cells were harvested by a rubber policeman, concentrated by centri­fugation (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 l. The antiproliferative effects were expressed as percentage of cel! numbers in control samples. White irradiation or IFNa. alone, moderately inhibited celi growth, the treatments, by IFNa. plus irradia-lrradiation, lzypertlzermia, inte1feron ,1 and catlz!'psi11 D 273 tion, and in particular, when IFNn was cornbin­ed with irradiation and hypertherrnia, the anti­proliferative effect was rnarkedly enhanced. Table l. Celi numbers (x105 HEF cells) after combined treatment with irracliation ancl/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 Interferona 6,6 ± 0,2 8,7± 1,7 61 43 lrradiaton + Interferona 3,7 ±0,1 4,0±0,4 34 20 I rracliation + Interferona+ 1,8±0,1 1,9±0,2 16 9 Hyperthennia The combined effects of IFNn, hypertherrnia and irradiation on celi proliferation, expressed as the numbers of growing HEp2 celi popula­tion, are shown in Table 2. The antiproliferative effects were expressed as percentage of celi numbers in control samples. While irradiation or IFNn alone, moderately inhibited HEp2 celi growth, the treatmens by IFNn plus irradiation, and in particular when IFNn 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) celi lines following combined treatment by interferon n and by hyperthermia are shown in Table 3. Table 2. Celi numbers (x 105 HEp2 cells) after combin­ed treatment with irracliation and/or interferon a and/ or hyperthermia. Group of Incubation time Percent of treatment after treatment control 24h 96h 2411 96h Controls 16,4± 1,4 41,0 ± 4,2 lrradiation 12,0±0,9 18,6 ± 1,6 73 45 Interferon a 11,0±2,l 21,8 ± 4,0 67 53 Irradiation + Interferon a 8,0±1,1 l0,0 ±0,7 48 24 Irradiation + Interferon n + 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 n increased the levels of cathepsin D in both celi strains. These effects are more expressed in malignant cells (1.5), than in nonrnalignant cells (1.2). Interferon n 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 rnalignant (1.6) as in nonmalignant (1.8) cells. Combined application of ali three agents together was most effective in increasing the amounts of cathepsin tested. Most evident effects were achieved in both celi lines after combined treatment with ali three agents and again, with more expressed effects Table 3. Changes in the quantity of Cathepsin D in irracliatecl HEF ancl HEp2 cells following combinecl treatment. Group of HEFcells HEp2 cells treatment Cathepsin D T/C* Cathepsin D T/C* ng/mg proteins ng/mg proteins Controls l05 ± l1 562 ± 14 Irracliation 138 ±7 1,3 604 ± 12 1,1 Interferon n 127±9 1,2 875 ±22 1,5 1 rracliation + Interferon n 198 ±26 1,8 856 ± 31 1,6 Irracliation + Interferon n + 332 ± 16 3,1 1407 ± 53 2,5 Hyperthermia * T/C Treatecl/Control274 Ferle-Vidovic A. et a/. on the nomnalignant 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 meta­stasise 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 intra­cellular concentrations of cathepsin D, and the­refore could consequently enhance the potential of the tumor cells to infiltrate the neighbouring tissues or to metastasise. lf 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 l. Holzer H, Heinrich PC. Control of proteolysis.Ann Rev Biochem 1980; 40: 63-91.2. Korbelik M, Škrk J, Suhar A, Turk V. The roleof proteinases, interfcrons and hormones in proli­fcrative activities of nonmalignant and malignantcells. Neoplasma 1988; 35: 555-63.3. Schcr W, Scher BM, Waxman S. Proteases stimu­late mouse erythroleukemia celi differentiationand multiplication. Biochem Biophys Res Comm1982; 109: 348-54.4. Walker CG. Induclible DNA repah- systems. Ann Rev Biochem 1985; 54: 425-57.5. Leto G, Gebbia N, Ransa L, Tuminello FM.Cathepsin D in the malignant progression of neo­plastic diseases (Review). Anticancer Research 1992; 12: 235-40.6. Baron S, Tyring SK, Fleischmann WR Jr et al.The interfcrons: mechanisms of action and clinicalapplications. JAMA 1991; 266: 1375-83.7. Heron I, Berg K. The actions of interferon arepotentiated at elcvated temperatures. Nature 1978;274: 508-10.8. Anjum A, Fleischmann WR Jr. Effect of hyper­thermia on the antitumor actions of interfcrons.Journal of Biologica/ Regulators and HomeostaticAgents 1992. 6: 75-86.9. Park RI, Richtsmeier WJ. Hyperthermia effectson the growth of a laryngeal squamous celi carci­noma celi line treated with recombinant humaninterferons a. and y. Oto/ Laryngol-Head andSurgery 1989; 101: 542-8.10. Perez CA, Nussbaum G, Emami B., VongerichtenD. Clinical results of iradiation combined withlocal hyperthermia. Cancer 1983; 52: 1597.11. Ferle-Vidovic A, Kaštelan M, Petrovic D, SveticB, Škrk J, Gabrijelčič D, Turk V. Cytotoxicitypotentiation of irradiation and cytostatic - mcasur­cd by changes in quantity of intracellular proteina­ses. Proc. of the First Symp. of Croatian Rad.Protect. Ass., Zagreb, Croatia 1992; 68-71.12. Petrovic D, Fcrle-Vidovic A, Škrk J, Suhar A,Turk V. Effects of irradiation and THP-Adriamy­cin on the proteinase activity profiles in culturesV 79 cells. Radio/ Oncol 1993; 27: 44-8.

Changes in the quantity of cathepsin D in irradiated human cells following treatment with hyperthermia and interferon [alpha]

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Changes in the quantity of cathepsin D in irradiated human cells following treatment with hyperthermia and interferon [alpha]

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