Antiproliferative Effect of Retinoid Compounds on Kaposi's Sarcoma Cells Jacques Corbeil, * Eric Rapaport, * Douglas D
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Antiproliferative Effect of Retinoid Compounds on Kaposi's Sarcoma Cells Jacques Corbeil, * Eric Rapaport, * Douglas D. Richman, * * and David J. Looneyt Departments of *Pathology and WMedicine, University ofCalifornia San Diego, La Jolla, California 92093; and ISan Diego Veterans Affairs Medical Center, San Diego, California 92161 Abstract duction, differentiation, and immune response, and may con- tribute in limiting the growth of certain malignancies. Reti- A panel of retinoid compounds (tretinoin, isotretinoin, acitre- noids have been used in the treatment of a variety of tin, and R013-1470) were tested for inhibitory activity against malignancies, including KS, with mixed results (22-26). The Kaposi's sarcoma cell (KSC) cultures in vitro. Tretinoin was effects of retinoids are mediated by two families of nuclear found to be the most effective retinoid tested, inhibiting the retinoic acid receptors (27-30) (RAR-a, -A, -y and RxRa). growth of KSC in vitro while having no effect on the expression Retinoids have been shown to inhibit the growth of human of interleukin-6 and basic fibroblast growth factor, two impor- melanoma cell lines by downregulating IL-6 receptors (31), tant cytokines involved in KSC growth. Tretinoin also did not providing a rationale for the investigation of retinoid com- appear to downregulate the expression of receptors for these pounds as therapeutics in KS. Promising results have been ob- two cytokines. At low concentrations (10-9 M), acitretin and tained with topical monotherapy of KS with tretinoin (32). tretinoin selectively inhibited growth of early passage KSC. At In this laboratory, KS cell (KSC) cultures derived from higher concentrations (10'-10-' M), retinoid treatment in- pleural fluid and cutaneous or mucosal biopsies have been suc- duced a pattern of DNA degradation and morphological cessfully established, including KSC cell cultures from both changes in KSC characteristic of apoptosis (programmed cell HIV-positive and -negative individuals. To determine the re- death). The inhibitory activity of tretinoin on KSC growth was sponse of these KSC cultures to retinoids, KSC were treated decreased if human serum (but not fetal calf serum) was pres- with graded concentrations of a representative panel of reti- ent in the growth medium, and partially restored by removal of noids (tretinoin, isotretinoin, acitretin, and Rol 3-1470), and serum lipids. These data suggest that retinoids possess poten- inhibition ofproliferation and cytotoxicity was correlated with tial as therapeutic agents in Kaposi's sarcoma. (J. Clin. Invest. the effects of retinoid treatment on IL-6 and bFGF, and their 1994. 93:1981-1986.) Key words: Kaposi's sarcoma * retinoids receptors. * chemotherapy * apoptosis * AIDS Methods Introduction Cell culture. KSCl was obtained from a explant of a biopsy obtained Kaposi's sarcoma (KS)' is epidemic in HIV-infected individ- from a young HIV-l-negative homosexual male with KS principally uals. A substantial proportion ofhomosexual males with AIDS involving the trunk and upper extremities. KSC2, 3, and 4 were ob- ( 15-18%) experience clinical complications due to KS during tained from HIV-infected homosexual men by explant from biopsies the course oftheir disease ( 1-3). Autopsy series have shown an from cutaneous lesions of disseminated HIV-associated KS. All ex- in A plants were grown in D-valine (GIBCO BRL, Gaithersburg, MD) even higher prevalence (59-95%) this group (4, 5). grow- DMEM supplemented with 10% endothelial cell-conditioned medium ing body of evidence on the growth characteristics of KS cells and 1% Condimedam9 (Boeringer Mannheim Biochemicals, Indianapo- cultured in vitro indicates the importance of autocrine path- lis, IN). Human umbilical vein endothelial cells (HUVEC) were pur- ways in regulation of proliferation, including prominent roles chased at passage 2 from Clonetics (San Diego, CA). The smooth mus- played by IL-6, basic fibroblastgrowth factor(bFGF), Oncosta- cle cell line SKLMS- I and the fibroblast cell line CCD34Lu were ob- tin M, and PDGF, as well as the HIV-l tat protein (6-14). tained from the American Type Culture Collection (Rockville, MD). Therapy, including the use of vinca alkaloids, radiotherapy, For all studies, cells were passaged using an enzymatic preparation interferon-alpha, and others, alone and in combination, has capable of releasing the cells from the substratum (Passage-ease; Vec- been directed towards palliation ofboth cutaneous and visceral tec, Schenectady, NY) and grown in EGM (Clonetics) that contains disease ( 15-21 ). 2% fetal bovine serum supplemented with epidermal growth factor (10 ng/ml), hydrocortisone (1 tg/ml), and a bovine brain extract ( 12 Retinoids are pharmacologic compounds derived from vi- ug/ml) containing heparin (10 ,g/ml) and the antibiotics gentamicin tamin A (retinol), which plays a critical role in growth, repro- (50 ug/ml) and amphotericin-B (50 ng/ml). Alternatively, the cells were grown in Endothelial-serum-free media (SFM) supplied by GIBCO BRL for the thymidine uptake assay. Address correspondence to Dr. Jacques Corbeil, Department ofMedi- Chemicals. Tretinoin (all-trans-retinoic acid); isotretinoin ( 1 3-cis- cine 0679, University of California San Diego, La Jolla, CA 92093- retinoic acid); acitretin ([all-trans-9 (4-methoxy-2,3,6-trimethyl- 0679. phenyl)-3,7-dimethyl-2,4,6,8-nonatetraenoic acid] and RO13-7410 Receivedforpublication 4 August 1993 and in revisedform 18 Jan- (p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra methyl-2-naphthyl)-1- uary 1994. propenyl] benzoic acid) were obtained from Roche Products (Dee Why, Australia). The compounds were dissolved in DMSO at 10-2 M 1. Abbreviations used in this paper: bFGF, basic fibroblast growth fac- and diluted in the culture media at the final dilution used. The stock tor; HUVEC, human umbilical vein endothelial cells; KSC, Kaposi's solutions were stored at -70°C protected from light and oxygen. sarcoma cell; PI, propidium iodine. Apoptosis assay. Treated and controls cells were released from the substratum using Passage-ease and fixed in 30% ethanol in PBS. Pro- The Journal ofClinical Investigation, Inc. pidium iodine (PI) was added for 30 min at a concentration of 50 Volume 93, May 1994, 1981-1986 ,gg/ml in PBS containing 0.1% Triton X-100, 0.1 mM EDTA(Na)2, Inhibition ofKaposi's Sarcoma Cell Growth by Retinoids 1981 and RNAse at 50 Mg/ml (50 U/mg). The cells were washed twice in polymerase (Boehringer Mannheim Biochemicals), 200 MM of each PBS and analyzed in a FACS® (Becton Dickinson & Co., Mountain dNTP, 100 pmol of each primer, 10 mM Tris-HCl, pH 8.3, 50 mM View, CA) using a cell cycle analysis doublet discrimination proto- KCI, 1.5 mM MgCl2, and 0.1 mg/ml gelatin. The reaction mixture was col (33). overlaid with 100 Al mineral oil and subjected to an initial denaturation [3H] Thymidine uptake assay. Cells (KSC, fibroblasts, smooth step followed by 35 cycles of 30 s at 940C, 30 s at 550C, and 1 min at muscle cells, and HUVEC) were plated in 96-well plates (previously 72"C, with a final 7-min extension step at 720C. Aliquots ( 12 ,l) ofthe coated with fibronectin for KS and HUVEC) at a density of 3,000- PCR products were subjected to electrophoresis in a 1.2% agarose gel 5,000 cells/well. The cells were maintained in endothelial serum-free (FMC Bioproducts, Rockland, ME) with predigested lambda molecu- medium (GIBCO BRL) for the duration of the experiment. Controls lar weight markers and 0.5 Mg/ml ethidium bromide and visualized included endothelial-SFM, and endothelial-SFM with DMSO added to under UV light. the same final concentration present in each corresponding dilution of Quantification ofIL-6. IL-6 ELISA was performed on culture su- retinoid solution ( 14 mM DMSO for I0-5 retinoid dilutions, 140 ,M pernatants (stored at -800C) as described by the manufacturer (R&D DMSO for l0-7 retinoid dilutions, and 1.4 MM DMSO for l0-9 reti- Research, Minneapolis, MN). This assay uses a polyclonal capture noid dilutions). Acitretin, tretinoin, isotretinoin, Ro 13-7410, and antibody and monoclonal antibodies for detection (35). DMSO were diluted in endothelial-SFM to obtain concentrations of 10-5, 10-7, and 10 -9 M ofeach retinoid ( 14 mM, 140 MM, and 1.4IM Results DMSO, respectively), and 200 Ml was added to each well. [3H]- The effect of four retinoid compounds on the proliferative re- thymidine (specific activity, 20 mCi/ml) was added to a final concen- tration of2 MCi/well (0.2 MCi/ml for the HUVEC). Each experimental sponse of cultures of three KS cell strains and control cells condition was assayed in quadruplicate. After 2 d, the cells were rinsed (endothelial cells, smooth muscle cells, and fibroblasts) was once with M 199 and 200 Ml of a solution of trypsin-EDTA (GIBCO determined by using a standard [3H]thymidine uptake assay. BRL) was added to release the adherent cells from the matrix. The cells All of the compounds tested, with the exception of acitretin, were harvested using a Skatron harvester, and the radioactive signal inhibited the growth rate ofKSC I by 50-70% at I0-5 M (Fig. 1 was determined by placing the filters in 5 ml of Ready-Safe cocktail A). The retinoid effects were negligible at the lower concentra- (Beckman Instruments, Fullerton, CA) followed by counting in the tions of l0-7 and 10-9 M. The proliferation rates ofKSC2 (Fig. tritium channel of a scintillation counter (ICN Micromedic, Hunts- 1 B) and KSC4 (Fig. 1 E) were also reduced, but to a lesser ville, AL). The resulting counts for each experimental condition were extent, ranging from 20 to 90% depending on the compound the mean of The quadruplicates. percentage ofcontrol was obtained by and concentration assayed. The KSC3 culture (Fig. 1 C) was dividing the mean ofa condition by the value obtained for the DMSO control without retinoid.