Bradykinin stimulation does not induce intracellular ...

ONCOLOGY REPORTS 3: 1161-1163, 1996

Bradykinin stimulation does not induce intracellular Ca2+ elevation in cells from desmoid tumors GIUSEPPE CASSANO1, FRANCESCO SUSCA2, CLAUDIO LIPPE1 and GINEVRA GUANTI2'3 ^stituto di Fisiologia Generale, Université di Bari; 2Cattedra di Genetica Medica della, Facoltà di Medicina, Université di Bari; 3IRCCS De Bellis, Castellana, Bari, Italy Received July 22, 1996; Accepted September 9, 1996

Abstract. The intracellular mechanisms controlling cell proliferation in desmoid tumors (DT) are unknown. Bradykinin stimulated an increase in [Ca2+],- (monitored by the fura-2 fluorescence) in fibroblasts obtained from both the skin of a normal donor and the mesenter of a familial adenomatous polyposis (FAP) patient. Cells from DT of the same patient as well as those from another FAP patient failed to show the elevation of [Ca2+]; usually caused by bradykinin stimulation. Introduction Desmoid tumors (DT) are benign locally aggressive neoplasms arising from musculoaponeurotic structures. They occur as isolated lesions and in association with other abnormalities. Surgery is the mainstay of therapy for these lesions, when vital structures are not involved, but recurrence is the rule when desmoids are not completely removed. Although the incidence of DT in the general population is as low as 0.1% of all tumors and 3.5% of fibrous tumors, their estimated occurrence in familial adenomatous polyposis (FAP) patients ranges from 4% to 32% (1). Several causative factors for the development of DT have been proposed. Trauma, especially that of previous surgery, has been associated with the subsequent development of DT, while the spontaneous development in FAP patients has been frequently described. Bradykinin (Bk) is a key mediator of the body's response to trauma and is generated locally at the site of tissue injury by the proteolytic cleavage of kininogen by kallicrein (2). At a cell level Bk induces, also via calcium signaling (3), several responses and mitogenic stimulation (4). Interestingly, it has been previously reported that Bk inhibits tumor growth in vivo, and that intralesional injection

Correspondence to: Dr Giuseppe Cassano, Istituto di Fisiologia Generale, Université di Bari, Via Amendola 165/A, 70126 Bari, Italy Key words: familiar adenomatous polyposis, bradykinin, calcium signaling, desmoid tumor, cell growth

of Bk markedly reduced the growth of spontaneous mammary tumors (MM46) and sarcomas (Sa 180) in mice (5) as well as SV40 virus induced fibrosarcomas in hamsters (6). On the basis of these assumptions, we decided to investigate the effects of Bk on desmoid cells and fibroblasts from FAP patients. Materials and methods Fibroblasts from mesenter and cells from desmoid tumors were obtained from biopsy specimens taken at surgery, while skin fibroblasts from a forearm biopsy of a young healthy male. Cell cultures were grown in RPMI 1640 (ICN Flow) containing fetal calf serum 10%, L-glutamine 2 mM, streptomycin 100 p.g/ml, penicillin 100 U/ml. Cells were maintained at 37°C and under 5% C0 2 in humidified air until they were confluent. Once confluence was reached, cells were detached with a trypsin/EDTA solution (0.05%/0.02%) and seeded onto glass coverslips (11x14 mm). When cells achieved the near confluence stage (48-72 h before the experiment), the medium was replaced with RPMI without serum. To monitor the [Ca 2+ ] ; the dual-wave-length ratiometric dye fura-2 was used (7). Fibroblasts, grown on a glass coverslip, were incubated for 30 min (in 5% C0 2 ) in the culture medium containing fura-2 acetoxymethylester 6.7 u.M. The coverslip was rinsed twice by keeping it, 3 min, in an assay buffer containing (in mM): NaCl 140, KCl 5, CaCl2 2, MgS0 4 1.2, KH2 P0 4 1.2, glucose 6, Hepes 25 pH 7.4 (with NaOH). Finally the coverslip was inserted into a square 1 cm disposable plastic cuvette, using a specially designed holder. The side of the coverslip without cells was facing the excitation beam at a 60° angle. The solution into the cuvette was under continuous stirring; additions were made by microsyringes. By using a Shimadzu RF-5000, the ratio between the fluorescence emission (510 nm) values respectively caused by an excitation beam of 340 and 380 nm was measured; the emission and excitation band-widths were set respectively to 5 and 3 nm. In order to calibrate the fluorescence ratio values on [Ca2+]„ at the end of every experiment we consecutively added into the cuvette: a) 5 \i\ of ionomycin (from an ethanolic stock solution, 3.5 pJVI final concentration); b) 50 |il of EGTA 300 mM (pH 7.5 with Tris, 7.5 mM final

1162

CASSANO et al: CELLS FROM DESMOID TUMORS ARE INSENSITIVE TO BRADYKININ

Figure 1. [Ca2*],- response to bradykinin in fibroblasts from the skin of a normal donor.

Figure 2. [Ca2+], response to bradykinin in: A) fibroblasts from the skin of a donor not affected by desmoid tumors; B) cells from a desmoid tumor.

concentration), c) 50 \x\ of Tris 2.4 M (60 mM final concentration); this calibration procedure is showed in Fig. 1. The [Ca2+]; values were calculated according to the following equation: [Ca2+],. = Kd((F-Fmin)/(Fmax-F)) where Kd= 224, Fmi]1 the value measured at the end of the experiment, Fmax the value reached after ionomycin addition (7,8). Bradykinin (stored as a stock solution in acetic acid 5%), fura-2 acetoxymethylester (in dimethyl sulfoxide), ionomycin (in ethanol), thapsigargin (in ethanol) were purchased from Calbiochem; vasopressin was obtained from Sigma. Results Changes of [Ca2+]; in response to Bk were measured in skin fibroblasts of a normal donor, by monitoring fura-2 fluorescence. As shown in Fig. 1, Bk (final concentration = 100 nM) typically produces a biphasic response; [Ca2+]; rises abruptly to a peak, then it declines to a plateau above basal levels. Fig. 1 also shows the effect of consecutive additions of ionomycin, EGTA and Tris; this experimental manoeuvre makes the calculation of [Ca 2+ ] ; possible from the fluorescence ratio values, and it was performed with each cell-preparation used in our experiments. The results of such a calculation are reported in the panel A of Fig. 2. Identical results have already been described in human foreskin fibroblasts (3). The experimental procedure described in Fig. 1 was then repeated using fibroblasts from a mesenter and a desmoid tumor of a patient affected by FAP. In the fibroblasts from mesenter (not shown) the Bk stimulation induced an elevation of [Ca2+]; similar to that observed in fibroblasts from a donor not affected by DT. Surprisingly in DT cells from the same patient (Fig. 2B), Bk failed to induce an elevation of [Ca2+],-. On the other hand, when desmoid cells were treated with thapsigargin (final concentration = 5 (iM), Fig. 3B, and ionomycin (not shown), the expected change of [Ca 2+ ] ; was recorded. Finally vasopressin (final concentration = 20 mU/ml), another agonist acting via calcium signaling, induced modest

Figure 3. [Ca2+], response to thapsigargin in: A) fibroblasts from the skin of a donor not affected by desmoid tumors; B) cells from a desmoid tumor.

Figure 4. [Ca2+], response to vasopressin in cells from a desmoid tumor.

elevation of [Ca2+], shown in Fig. 4. These observations allow us to exclude that the negative response of DT cells to Bk was the consequence of a defective experimental setup or cell preparation. In order to confirm these results, DT cells from another FAP patient were used. In this case the Ca2+ response to Bk was again absent; moreover vasopressin did not elicit any Ca2+ flux either. The results of our experiments are summarized in Table I, where the mean values of [Ca2+],- in the absence of stimulation

ONCOLOGY REPORTS 3: 1161-1163, 1996

1163

Table I. Summary of [Ca 2+ ] ; and sensitivity of bradykinin.

[Ca2+]; (nM)

[Ca2+]; after Bk

Fibroblasts from the skin of a normal donor

85+54(11)

IT (12)

Fibroblasts from the mesenter of the FAP patient 1

62±26 (5)

Cells from desmoid tumor of the FAP patient 1

65±40 (9)

1r (3) = (10)

Cells from desmoid tumor of the FAP patient 2

82±36 (6)

= (5)

The [Ca2+],- values are means +SD; the mean of all values was 75±44 nM. In parenthesis the number of observations.

are also reported; basal [Ca2+],- are not different in the four types of cells we used. Discussion Our experiments demonstrate that cells from DT of FAP patients did not show the elevation of [Ca2+],- usually caused by Bk stimulation, whereas mesenteric fibroblasts, from one of these patients, showed a positive response identical to normal control fibroblasts. The two possible mechanisms, actually under experimental evaluation, underlying this differential action of Bk are: i) the absence or improperly working Bk receptors; ii) a defect in the calcium signaling pathway. More intriguing, and at the moment without answer, are the following questions: i) is the unregulated growth of D T the cause or the effect of the defect in calcium signaling? ii) is the sensitivity to Bk stimulation peculiar to all DT or just to DT occurring in FAP patients? We are looking for cells from DT of non polyposis patients to answer the second question and to verify the possible involvement of the APC (adenomatous polyposis coli) gene product. The unregulated growth of DT cells and their insensitivity to Bk suggest that Bk can act as negative modulator of cell proliferation. The inhibitory effect of Bk on DNA synthesis has been previously reported in human gingival fibroblasts (9) and in rat k i d n e y cells ( 1 0 ) . E x p e r i m e n t s are in p r o g r e s s to investigate the role of Bk treatment in the regulation of DNA synthesis in DT cells. Acknowledgments Thanks are due to Professor M.L. Brandi (Department of Clinical Physiopathology, University of Florence, Italy) for providing the second desmoid tumor cell line. This work was supported by grants from M U R S T 4 0 % and 60%, AIRC, C N R P F A C R 0 94.1142.

References 1. Klein WA, Miller HM, Anderson M and DeCosse JJ: The use of indomethacin, sulindac, and tamoxifen for the treatment of desmoid tumors associated with familial polyposis. Cancer 60: 2863-2868, 1987. 2. Worthy K, Figueroa CD, Dieppe PA and Bhoola KD: Kallikreins and kinins: mediators in inflammatory joint disease? Int J Exp Pathol 71:5 S7-601, 1990. 3. Byron KL, Babnigg G and Villereal ML: Bradykinin-induced Ca2+ entry, release, and refilling of intracellular Ca2+ stores. J Biol Chem 267: 108-118, 1992. 4. Aaronson SA: Growth factors and cancer. Science 254: 1 Mo­ ll 50, 1991. 5. Mashiba H and Matsunaga K: Tumor-inhibitory effect of intralesional injection of bradykinin and immunostimulants in mice. Cancer Lett 29: 177-182, 1985. 6. Koppelmann LE, Moore TC and Porter DD: Increased plasma kallikrein activity and tumor growth suppression associated with intralesional bradykinin injection in hamsters. J Pathol 126: 110, 1978. 7. Grynkiewicz G, Poenie M and Tsien RY: A new generation of Ca indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985. 8. Kao JPY: Practical aspects of measuring [Ca2+] with fluorescent indicators. Methods Cell Biol vol. 40: 155-181, 1994. 9. McAllister B, Leeb-Lundberg F and Olson MS: Bradykinin inhibition of EGF- and PDGF-induced DNA synthesis in human fibroblasts. Am J Physiol 265: C477-C484, 1993. 10. Van Zoelen E, Peters PH, Afink GB, Van Genesen S, De Roos AD, Van Rotterdam W and Theuvenet PR: Bradykinin induced growth inhibition of normal rat kidney (NRK) cells is paralleled by a decrease in epidermal-growth-factor receptor expression. Biochem J 298: 335-340, 1994.