17

Pharmacological Research, Vol . 25, Supplement 1, 1992 THE ANTIPROLIFERATIVE EFFECT OF SURAMIN ON THE CANCER CELL LINE SW-13 Is MEDIATED BY THE INHIBITION OF TRANSFORMING GROWTH FACTOR [i1 (TGF-(31) Danesi R, Agen C, Bernardini N, Costa M, Del Tacca M Istituto di Farmacologia, Facolty di Medicina e Chirurgia, University degli Studi di Pisa, Via Roma 55, 56100 Pisa Key words: Suramin - TGF-131 - Adrenal cancer - Cell proliferation - Antisense oligonucleotides

Suramin is a new anticancer agent with structural and functional properties similar to heparin . The inhibition of the function of a variety of heparin-binding growth factors (including FGF and TGF-[31) and enzyme systems represents the principal mechanism of action of the drug which has shown promising therapeutic effect in some malignancies (1) . TGF-(31 is a disulfide-linked homodimer which belongs to a family of structurally related polipeptides that regulate cell growth and differentiation (2) . TGF-R 1 stimulation of target cells results in the increased expression of protooncogenes such as c-sis, c-my c, and

c fos;

recent studies have provided evidence for both G-protein-

dependent and -independent signaling as the mediators of TGF-(31 action (3) . The present study evaluated the relationship between TGF-p1 gene expression and the antiproliferative effect of suramin on the human adrenal cancer cell line SW-13 . The colony formation assay was used to evaluate the cytotoxic effect of suramin . SW13 cells were plated in 6-well dishes containing L-15 medium and treated with suramin (50-300 .tg/mL) ; after 6 days medium was replaced and the resulting colonies were counted after additional 10 days. The suramin concentration resulting in 50% inhibition of colony formation with respect to the untreated controls (IC50) was calculated . For the Northern blot-hybridization analysis total RNA was extracted from cells by using the guanidinium isothiocyanate/CsCl method . Samples of 15 µg were fractionated on a 1 .1% agarose/formaldehyde gel, stained with ethidium bromide and visualized for equivalent loading of RNA . The following steps were performed according to the supplier of the nylon membranes (Schleicher & Schuell) . Membranes were exposed to Kodak XAR-5 film at -70 °C for autoradiography. The TGF-R 1 DNA probe (1 .05 kb fragment in the EcoRI site of pSP64) was labeled with [a- 32P]dCTP (NEN-Dupont) by the random-primer method (Pharmacia) . The mytogenic activity of TGF-[il was evaluated on confluent SW-13 cells treated with TGF-(31 (0 .1-100 ng/mL) alone or with the addition of suramin (300 µg/mL) . After 30 h, the cells were pulsed with 1 jCi/mL of 5-bromo-2'-deoxy[l', 2'-3H]uridine ([ 3H]BrdU, NEN-Dupont) for 5 h ; then they were harvested onto glass fiber filters with a PHD cell harvester and the radioactivity counted with a Packard 2000 CA Tri-carb liquid scintillation counter . Antisense experiments were performed on 60% confluent cells plated in 96-well dishes . Cells were treated with antisense and random TGF-(31 phosphorothioate oligonucleotides (20

1043-6618/92/2510017-02/$03 .00/0

© 1992 The Italian Pharmacological Society

18

Pharmacological Research, Vol . 25, Supplement 1, 1992

µM each) synthesized on a Applied Biosystems model 381 A DNA synthesizer ; cells were labeled with 1 µCi/mL of [ 3 H]BrdU for 5 h, 18 h after the addition of oligonucleotides and the incorporated radioactivity was measured as reported above . The proliferation of the SW-13 cell line was inhibited by suramin in a dosedependent manner (Fig . 1) . The IC50 was 75 tg/mL, a concentration that is clinically achievable in cancer patients . The Northern blot-hybridization analysis of total RNA extracted from the SW- 13 cell line demonstrated a detectable TGF-p1 gene message ; treatment with suramin resulted in a striking decrease of the level of expression of the TGF-p1 mRNA . TGF-p1 treatment stimulated the [3 H]BrdU incorporation by the 250% compared to untreated cells ; suramin markedly reduced its mytogenic effect . A low concentration (20 µM) of the antisense oligonucleotide was able to inhibit the [3H]BrdU incorporation by the cells by the 80% (Fig. 2) while the random sequence was almost devoided of such effect (Fig . 2). X

40

a 0 ~_ 8 o m30 2 i =

s:.

20-

20

0 0

100 200 Suramin, µg/ml

300

Fig . 1 . Effect of a 6-days exposure to suramin on the colony formation of the SW-13 cell line

Control

TGF- 431 AS

TGF-

01

RD

Fig . 2 . Effect of TGF-(31 antisense (AS) and random (RD) oligonucleotides on DNA synthesis in the SW-13 cell line

The data of the present study suggest that TGF-p 1 is involved in the proliferation of the adrenal cancer cell line SW- 13 and that the cell growth inhibition by suramin is mediated, at least in part, by the inhibition of the mitogenic activity of TGF-p l . 1 . La Rocca RV, Stein CA, Myers CE. Suramin : prototype of a new generation of antitumor compounds . Cancer Cells 1990;2 :106-115 2 . Massague J . The TGF-beta family of growth and differentiation factors . Cell 1987 ;49 :437-438 3 . Howe PH, Bascom CC, Cunningham MR, Leof EB . Regulation of transforming growth factor-p1 action by multiple transducing pathways : evidence for both G protein-dependent and -independent signaling . Cancer Res 1989;49 :60246031 This work was supported in part by AIRC (Milano, Italy) and CNR (Rome, Italy), P. S . "Interazioni reciproche fra sistema endocrino, nervoso e immunitario"

The antiproliferative effect of suramin on the cancer cell line SW-13 is mediated by the inhibition of transforming growth factor beta 1 (TGF-beta 1).

17 Pharmacological Research, Vol . 25, Supplement 1, 1992 THE ANTIPROLIFERATIVE EFFECT OF SURAMIN ON THE CANCER CELL LINE SW-13 Is MEDIATED BY THE IN...
252KB Sizes 0 Downloads 0 Views