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Receptor tyrosine kinase inhibition suppresses growth of pediatric renal tumor cells in vitro
Receptor Tyrosine Kinase Inhibition Suppresses Growth of Pediatric Renal Tumor Cells In Vitro By Shalizeh Naraghi, Sami Khoshyomn, Joseph A. DeMattia, and Dennis W. Vane Burlington, Vermont
Purpose: Children who undergo standard therapy for renal tumors are at an increased risk for treatment sequelae such as congestive heart failure, abnormal trunk development, and secondary malignancies. Therefore, research on the use of novel chemotherapeutic agents with fewer side effects is justified. Recent experimental evidence suggests that growth factor receptors such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) play an important role in growth and development of pediatric renal tumors especially that of Wilms’ tumor. In this study we investigated the effects of genistein, AG1478, and AG1295, from the class of growth factor receptor tyrosine kinase (GFR-TK) inhibitors, on proliferation and colonigenic growth of 2 pediatric renal tumor cell lines.
(P ⫽ .01 and 5E-06, 2-tailed t test, respectively) and that of SK-NEP-1 by 44% and 74% (P ⫽ .0001 and 9.9E-07). The mean percent growth inhibition at the above doses was 57% ⫾ 7.9% and 96% ⫾ 0.2% for G-401, and 47% ⫾ 11.2% and 60% ⫾ 2.7% for SK-NEP-1. AG1478 at concentrations of 1,000 and 5,000 mol/L inhibited the colonigenic growth of G401 by 75% and 78% (P ⫽ .0005 and 7.38E-06, respectively) and that of SKNEP-1 by 19% and 40% (P ⫽ .02 and .0001). The percent growth inhibition at the mentioned concentrations for G-401 were 53% ⫾ 9.3% and 63% ⫾ 6.3%, and for SK-NEP-1 were 55% ⫾ 14.5% and 65% ⫾ 20.1%, respectively. AG1295 did not appear to be as effective as AG1478.
Methods: The authors studied the effect of genistein (broadspectrum GFR-TK inhibitor), AG1478 (EGFR-specific GFR-TK inhibitor), and AG1295 (PDGFR-specific GFR-TK inhibitor) on proliferation and colonigenic growth of rhabdoid tumor of the kidney and Wilms’ tumor cell lines: G-401 and SK-NEP-1, respectively. The effect of genistein at concentrations of 0 to 200 µmol/L, and AG1478 and AG1295 at 0 to 10,000 mol/L were tested on proliferation by using a growth inhibition assay. Viable cell counts at each concentration were obtained by hemocytometer and trypan blue exclusion, and percent growth inhibition was calculated based on control cultures at the same time-point. As a measure of colonigenic survival, the percent inhibition of colony formation in drug-treated dishes was calculated based on the number of colonies (⬎50 cells) in control dishes.
Conclusions: This is the first experimental study on the use of GFR-TK inhibitors as a potential treatment for pediatric renal tumors. GFR-TK inhibitors such as genistein occur naturally in soybean foods and have been shown to reach therapeutic levels in blood after consuming a soybean-based diet. Considering the significance of growth factor receptor activity in Wilms’ tumor development, inhibition of GFR-TKs should be investigated as effective and potentially nontoxic adjunctive treatment for this childhood tumor. Furthermore, GFR-TK inhibitors may offer an effective alternative to the treatment of commonly fatal rhabdoid tumor of the kidney in children. J Pediatr Surg 35:884-890. Copyright 娀 2000 by W.B. Saunders Company.
Results: Genistein at concentrations of 25 and 50 µmol/L inhibited the colonigenic growth of G-401 by 37% and 79%
INDEX WORDS: Pediatric renal tumors, tyrosine kinase receptor inhibitors.
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this once uniformly lethal disease. Still, children who undergo standard therapy for Wilms’ tumor are at an increased risk for treatment sequelae such as congestive heart failure, abnormal trunk development, and secondary malignancies.1-3 In addition, 11% of the Wilms’ tumor patients present with an anaplastic variant of the disease that, despite advances in therapy, is still associated with high rates of recurrence and death.1 Rhabdoid tumor of the kidney is an uncommon renal tumor of childhood that is associated frequently with highly aggressive behavior, resistance to multiagent chemotherapy, and fatal metastasis.4 This, together with the late adverse effects of cancer treatment in children, justifies research on the future use of novel and perhaps minimally toxic chemotherapeutic agents. Increased expression of growth factor receptors (GFRs)
ILMS’ TUMOR is the most common primary malignant renal tumor in children, accounting for about 6% to 7% of all pediatric neoplasms. Advances in surgical techniques and postoperative care together with the sensitivity of the tumor to radiation and chemotherapy has led to a dramatic improvement in the prognosis of From the Department of Surgery, University of Vermont, Burlington, VT. Presented at the 1999 Annual Meeting of the Section on Surgery of the American Academy of Pediatrics, Washington, DC, October 8-10, 1999. Address reprint requests to Dennis W. Vane, MD, Department of Surgery, University of Vermont, Burlington, VT 05401. Copyright 娀 2000 by W.B. Saunders Company 0022-3468/00/3506-0015$03.00/0 doi:10.1053/js.2000.6907 884
Journal of Pediatric Surgery, Vol 35, No 6 (June), 2000: pp 884-890
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including epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and insulin-like growth factor receptor (IGFR) in human epithelial tumors is correlated with poor prognosis and short survival times in patients with carcinoma of the breast,5 lung,6 nasopharynx and mouth,7,8 bladder,9 esophagus and gastrointestinal tract,10-13 pancreas cancer,14,15 endometrium,16 and thyroid gland.17 In glioblastomas and sarcomas (bone and soft tissue) as nonepithelial tumors, growth factor receptor overactivity is linked to shorter survival and time to recurrence after treatment.18,19 Recent experimental evidence suggests that growth factor receptors such as EGFR and PDGFR play an important role in the growth and development of embryonic cancers such as osteosarcoma and Wilms’ tumors.20-22 It is the guiding hypothesis of this study that inhibiting the action of these receptors by suppressing their intrinsic tyrosine kinase activity can block the mitotic and colonigenic growth of pediatric renal tumors. To clarify this, we have used genistein and tyrophostins (AG1478 and AG1295) from the class of receptor tyrosine kinase (RTK) inhibitors.23 These agents have been shown to have no in vivo toxicity at effective therapeutic doses in a number of neoplastic and inflammatory diseases and potentially could be used as an alternative therapy for Wilms’ tumor or rhabdoid tumor of the kidney. MATERIALS AND METHODS For growth inhibition in this study 2 commercially available human renal tumor cell lines, G-401 and SK-NEP-1, from American Type Culture Collection (ATCC) were used. G401 line was derived from the rhabdoid tumor of the kidney of a 3-month-old boy and SK-NEP-1 line originates from cells of a malignant pleural effusion in a 25-year-old female patient with Wilms’ tumor.
Effect of Genistein, AG1478, and AG 1295 on Tumor Monolayer Cell Growth Cryopreserved G-401 and SK-NEP-1 cells were thawed rapidly and propagated in 75-cm tissue culture flasks (Falcon, Franklin Lakes, NJ) containing complete media. G-401 and SK-NEP-1 cells were grown in modified McCoy’s 5a media with L-glutamine supplemented with 10% and 15% heat-inactivated newborn calf serum, respectively, and 1% antibiotic-antimycotic solution (Gibco BRL, Grand Island, NY) under standard tissue culture conditions (100% humidity, 95% air, and 5% CO2). Subconfluent monolayers of cells were trypsinized and 1 ⫻ 105 were seeded into each well of a 12-well tissue culture plate (Falcon, Lincoln Park, NJ). A 0.1 mol/L stock solution of genistein (Calbiochem, La Jolla, CA) in DMSO (Sigma, St Louis, MO) was diluted in complete growth medium as described above to reach final concentrations of 10, 25, 50, 100 and 200 µmol/L. The concentration of DMSO in control media and at 200 µmol/L genistein concentration was 0.2%. Similarly, 0.01 mol/L stock solutions of tyrphostins AG1478 and AG1295 (Calbiochem, La Jolla, CA) in DMSO were diluted in complete growth media as previously described to reach concentrations of 100, 500, 1,000, 5,000, and 10,000 mol. The concentration of DMSO in control media and at 10,000 mol
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tyrphostin was 0.1%. Media containing each tested drug at abovementioned concentrations were added to each well 24 hours after plating of cells and were replenished every 24 hours with fresh complete medium containing the studied drug for 4 days. Cells were then harvested by trypsinization at 5 days, and viable cell counts were obtained by hemocytometer and trypan blue exclusion. Cell counts at each concentration were performed in triplicates. The percent growth inhibition was calculated based on control cultures using medium containing DMSO at the same time point.
Effect of Genistein, AG1478, and AG1295 on Colonigenic Growth of Tumor Cell Lines Cell suspensions of G-401 and SK-NEP-1 were incubated at the above doses of genistein, AG1478 and AG1295 separately for 24 hours. Next SK-NEP-1, and G-401 cells were centrifuged and washed in D-PBS, and seeded at a density of 1,000 cells in enriched complete medium (20% heat inactivated new born calf serum) in 60-mm culture dishes. A solid culture substrate was used here instead of semisolid agarose because these cells do not form colonies in agarose. G-401 cells form anchorage-dependent colonies and, SK-NEP-1 cells form nonadhering spheroids. The cultures were maintained under standard tissue culture conditions for at least 10 days. The number of colonies containing more than 50 cells in diameter were then counted at each drug concentration in each dish. The percent inhibition of colony formation (as the measure of inhibition of colonigenic survival) in drug-treated dishes was calculated based on the number of colonies (⬎50 cells in diameter) in control dishes.24
RESULTS
As explained earlier, effects of the receptor tyrosine kinase inhibitors genistein, AG1478 and AG1295 on renal tumor cell lines G-401 and SK-NEP-1 were measured in terms of inhibition of monolayer cell growth and colony formation on a solid substrate. These cells did not form colonies in 0.5% agarose, but formed spheroids and adhering colonies on a solid surface. Effect of Genistein on Tumor Monolayer Cell Growth and Colonigenic Growth At the doses of 25 and 50 µmol/L genistein appeared to suppress monolayer cell growth of tumor cells by 57% ⫾ 7.9% and 96% ⫾ 0.2% for G-401, and 47% ⫾ 11.2% and 60% ⫾ 2.7% for SK-NEP-1. Genistein inhibited colony formation of G-401 at doses of 25 and 50 µmol/L by 37% and 79% (P ⫽ .01 and 5E-06, respectively; 2-tailed t test) and that of SK-NEP-1 by 44% and 74% (P ⫽ .0001 and 9.9E-07, respectively; Figs 1, 2, 3A-C, 4A-C). Effect of AG1478 and AG1295 Tumor Monolayer Cell Growth and Colonigenic Growth AG1478 (EGFR-specific GFR-TK inhibitor) at concentrations of 1,000 and 5,000 mol/L inhibited the monolayer growth of tumor cells by 53% ⫾ 9.3% and 63% ⫾ 6.3% for G-401 and 55% ⫾ 14.5% and 65% ⫾ 20.1% for SK-NEP-1, respectively. AG1295 (PDGFR-specific GFR-TK inhibitor), however, did not appear to be as effective (Fig 5).
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Fig 1. Effect of genistein on monolayer cell growth of G-401 and SK-NEP-1 lines.
AG1478 at the above concentrations suppressed the colony formation of G-401 cells by 75% and 78% (P ⫽ .0005 and 7.38E-06, respectively; 2-tailed t test) and that of SK-NEP-1 by 19% and 40% (P ⫽ .02 and .0001). AG1295 at above concentrations did not significantly inhibit colony formation of G-401 cells: 9% and 7%, respectively (P ⫽ .41 and P ⫽ .66). AG1295 was similarly ineffective on SK-NEP-1 cells (Fig 6). DISCUSSION
Role of Growth Factor Receptor Tyrosine Kinases in Clinical Tumor Progression Activation of a growth factor receptor (GFR) such as EGFR, through the binding of its ligands (EGF or transforming growth factor [TGF-␣]), results in the induction of the receptor’s intrinsic tyrosine kinase (RTK) activity.25,26 This leads to autophosphorylation of intracellular tyrosine residues on the receptor, followed by tyrosine phosphorylation of various signaling proteins27 that lead to a series of cellular activities including proliferation and migration.28-30 As discussed earlier, increased expression of growth factor receptors including EGFR, PDGFR, and insulinlike growth factor receptor (IGFR) in human epithelial tumors is correlated with poor prognosis and short survival times in patients.5-17 In glioblastomas and sarco-
Fig 2. Effect of genistein on colony formation of G-401 and SK-NEP-1 cell lines.
Fig 3. SK-NEP-1 spheroids at 3 genistein concentrations. (A) 0 mol/L, (B) 25 mol/L, and (C) 50 mol/L. (Original magnification, ⴛ25.)
mas (bone and soft tissue) as nonepithelial tumors, growth factor receptor over activity is linked to shorter survival and time to recurrence after treatment.18,19 Intercepting the growth factor receptor activation cascade by using GFR-TK inhibitors has been, therefore, recognized as a target for anticancer drug design.23 Reports on the experimental in vitro and in vivo (animal experiments) use of GFR-TK inhibitors have shown encouraging results in blocking growth and invasiveness of a number of tumors such as breast cancer, colorectal carcinoma, glioblastoma multiforme, and leukemia.31-37
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Fig 5. Effect of AG1478 and AG1295 on monolayer cell growth of G-401 and SK-NEP-1 lines.
differentiation and growth of metanephric blastema has been described, the role of agents that control and regulate this process is not understood completely. It is, however, known that this process is coordinated by the timed and sequential interaction between the growth factors such as IGF, EGF, TGF-␣ and PDGF and their receptors. Therefore, loss of regulation of growth factor– mediated development and growth is believed to retard kidney differentiation and result in neoplastic transformation of metanephric blastema and formation of Wilms’ tumor.22,38 The origin of rhabdoid tumor of the kidney, however, is less certain. Previous findings have indicated that this tumor may arise from the metanephric blastoma as a result of a germline mutation. This suggestion is based on the observation that rhabdoid tumor commonly occurs together with primitive neuroectodermal tumor of the brain.39 So far the only growth factors implicated in the growth and development of rhabdoid tumor have been IGF I and II.40 The exact nature of their involvement remains unclear. Tumor Treatment Using Receptor Tyrosine Kinase Inhibitors
Fig 4. G-401 colonies at 3 genistein concentrations. (A) 0 mol/L, (B) 25 mol/L, and (C) 50 mol/L. Colonies were fixed using methyl alcohol and stained with methylene blue. (Original magnification, ⴛ40.)
Recent reports on the experimental in vivo use of selective RTK inhibitors have shown dramatic results in curing B-cell leukemia32 and preventing breast cancer.33,34 The most anticarcinogenic and antiproliferative group of these drugs, the isoflavones genistein and daidzein, occur naturally in soybeans and soy-protein
Growth Factor Receptors and The Biology of Wilms’ Tumor and Rhabdoid Tumor of Kidney Wilms’ tumor cells originate from the undifferentiated mesenchymal cells of metanephric blastema. Metanephric blastema, also called metanephrogenic mesenchyme, is that part of the mesoderm of the embryonic kidney that gives rise to the excretory tubules of the metanephros. Metanephros is the most caudally located part of the embryonic renal excretory organ, which will eventually serve as the permanent kidney in humans. Although the
Fig 6. Effect of AG1478 and AG1295 on colony formation of G-401 and SK-NEP-1 cell lines.
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foods41 and have been shown to reach effective levels in blood after consuming a soybean-based diet.42 Genistein is a potent and relatively specific inhibitor of receptor tyrosine kinase activity with a wide range of inhibiting concentration (IC)50 values (2.6 to 79 µmol/L) for proliferation of human tumor lines in vitro.34,43 Obtained through the diet, genistein can inhibit in vitro angiogenesis (endothelial cord formation inhibited at an IC50 of 5 µmol/L), and endothelial cell proliferation (IC50 of 150 µmol/L) stimulated by basic fibroblast growth factor (bFGF).44 Genistein inhibits plasminogen activator (PA) and PA-inhibitor-1 in vascular endothelium, which play a role in tumor angiogenesis.44 Genistein also inhibits the activity of topoisomerase II in vitro with IC50 values around 111 µmol/L.43 Previous experimental work has shown that genistein can block invasiveness of glioblastoma and breast cancer lines in organotypic coculture systems in vitro.31,35 Similar effects have been reported for synthetic RTK inhibitors such as tyrphostins as well.31,36,45 Tyrophostins are a group of synthetic simple organic compounds that specifically inhibit RTKs and not other protein kinases, by competing for the substrate binding site and not the ATP binding site (both are located on the intracellular domain), which is a common structural feature among all protein kinases. These compounds are, therefore, more potent, selective, and nontoxic inhibitors of RTKs than isoflavones (genistein and daidzein).23 For example, the tyrphostin AG1478 is a very potent and specific inhibitor of EGFR-TK activity (IC50 ⫽ 0.003 µmol/L) as compared with PDGFR-TK activity (IC50 ⬎ 100 µmol/L).46 The opposite relationship between effects exists for another member of this family; AG1295 (IC50 for PDGFR-TK inhibition is 0.5 µmol/L v little to no effect on EGFR-TK activity).46 Animal experiments using tyrphostins have shown that these compounds are nontoxic at up to 30 times their effective dose in vivo.47 In this study we examined the effect of the abovementioned RTK inhibitors on monolayer cell growth and colonigenic growth of 2 human renal tumor cell lines. Our results showed significant suppression of both proliferation and colony formation of the G-401 and SKNEP-1 lines by the EGFR selective RTK inhibitors genistein and AG1478 but not the PDGFR-specific AG1295. This suggests that perhaps EGFR signaling plays a role in the growth of these 2 tumor cell lines. Antitumor effects for RTK inhibitors have been shown in other childhood tumors such as neuroblastoma and osteosarcoma.48,49 Genistein suppresses the growth of neuroblastoma cell lines in vitro by inducing apoptosis and differentiation48 and blocks growth factor–induced migration of tumor cells in an osteosarcoma cell line.49 The significant effect of genistein on tumor cell growth in our study may be attributed to inhibition of RTK or
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topoisomerase II activity. Our preliminary results from mechanistic studies have so far shown that genistein at concentrations up to 50 µmol/L does not inhibit tumor cell growth by inducing apoptosis. As mentioned earlier, typical IC50 concentrations for topoisomerase activity as reported in the literature are greater than 100 µmol/L.43 Further experimental studies are still necessary to clarify the mechanism of action of these drugs. There have been to date no reports on any in vitro or in vivo use of RTK inhibitors as potential treatment for childhood renal tumors. The long-term goal of such experimental studies is to eventually proceed to animal experiments and human patient trials. In this connection the use of soybean isoflavones, such as genistein and daidzein, is particularly attractive because soy protein formulae have been widely used to feed infants with cow milk allergy since the early 1920s.50 A number of studies have shown that soy-based diets in full-term infants are well tolerated and have no effects on weight gain.51 Although bioavailability of soymilk isoflavones in children is still under investigation,52 a series of human trials has shown isoflavone (genistein and daidzein) plasma levels of up to 4 µmol/L after oral doses of 2 mg/kg body weight as a part of soymilk formulations in adults.42 Plasma isoflavone concentrations in 4-month-old infants fed soy-based formula have been reported to be between 3 and 6 µmol/L.53 Such low plasma concentrations may not justify the use of oral isoflavones in its current form as single-agent chemotherapy. There is, however, emerging evidence that even at a low dose there may be a significant synergistic antiproliferative effect with standard chemotherapy for some tumors.54,55 The significance of this for pediatric tumors such as Wilms’ tumor may be a reduction in the current dose recommendations for chemotherapy and radiation therapy without compromising their effectiveness but perhaps reducing the devastating treatment sequelae. Furthermore, isoflavones may offer a new therapeutic alternative where present multidrug chemotherapy has achieved little in treatment of rhabdoid tumor of the kidney in children. REFERENCES 1. Green DM, D’Angio GJ, Beckwith JB, et al: Wilms tumor. CA Cancer J Clin 46:46-63, 1996 2. DeLaat CA, Lampkin BC: Long-term survivors of childhood cancer: Evaluation and identification of sequelae of treatment. CA Cancer J Clin 42:263-282, 1992 3. Meadows AT, Baum E, Fossati-Bellani F, et al: Second malignant neoplasms in children: An update from the Late Effects Study Group. J Clin Oncol 3:532-538, 1985 4. Weeks DA, Beckwith J, Mierau GW, et al: Rhabdoid tumor of kidney. Am J Surg Pathol 13:439-458, 1989 5. Rudland PS, Fernig DG, Smith JA: Growth factors and their receptors in neoplastic mammary glands. Biomed Pharmacother 49:389399, 1995 6. Occleston NL, Walker C: Production of multiple growth factors
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26. Hunter T: The epidermal growth factor receptor gene and its product. Nature 311:414-416, 1984 27. Schlessinger J, Ullrich A: Growth factor signaling by receptor tyrosine kinases. Neuron 9:383-391, 1992 28. Aaronson SA: Growth factors and cancer. Science 254:11461153, 1991 29. Barrandon Y, Green H: Cell migration is essential for sustained growth of keratinocyte colonies: The roles of transforming growth factor-alpha and epidermal growth factor. Cell 50:1131-1137, 1987 30. Rheinwald JG, Green H: Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 265:421-424, 1977 31. Penar PL, Khoshyomn S, Bhushan A, et al: Inhibition of epidermal growth factor receptor-associated tyrosine kinase blocks glioblastoma invasion of brain. Neurosurgery 40:141-151, 1997 32. Uckun FM, Evans WE, Forsyth CJ, et al: Biotherapy of B-cell precursor leukemia by targeting genistein to CD19-associated tyrosine kinase. Science 267:886-891, 1995 33. Barnes S, Grubbs C, Setchell KD, et al: Soybeans inhibit mammary tumors in models of breast cancer. Prog Clin Biol Res 347:239-253, 1990 34. Barnes S: Effect of genistein on in vitro and in vivo models of cancer. J Nutr 125:777S-783S, 1995 35. Parmar VS, Jain R, Sharma SK, et al: Anti-invasive activity of 3,7-dimethoxyflavone in vitro. J Pharm Sci 83:1217-1221, 1994 36. Miyaji K, Tani E, Shindo H, et al: Effect of tyrphostin on cell growth and tyrosine kinase activity of epidermal growth factor receptor in human gliomas. J Neurosurg 81:411-419, 1994 37. Yanagihara K, Ito A, Toge T, et al: Antiproliferative effects of isoflavones on human cancer cell lines established from the gastrointestinal tract. Cancer Res 53:5815-5821, 1993 38. Hammerman MR: Growth factors in renal development. Semin Nephrol 15:291-299, 1995 39. Knudson AG: Introduction to genetics of primary renal tumors in children. Med Pediatr Oncol 21:193-198, 1993 40. Suzuki A, Ohta S, Shimada M: Gene expression of malignant rhabdoid tumor cell lines by reverse transcriptase-polymerase chain reaction. Diagn Mol Pathol Dec 6:326-332, 1997 41. Murphy PA: Phytoestrogen content of processed soybean products. Food Technol 36:62-64, 1982 42. Xu X, Wang H, Murphy PA, et al: Diadzein is a more bioavailable soymilk isoflavone than is genistein in adult women. J Nutr 124:825-832, 1994 43. Peterson G: Evaluation of the biochemical targets of genistein in tumor cells. J Nutr 125:784S-789S, 1995 44. Fotsis T, Pepper M, Adlercreutz H, et al: Genistein, a dietaryderived inhibitor of in vitro angiogenesis. Proc Natl Acad Sci U S A 90:2690-2694, 1993 45. Klemke RL, Yebra EM, Bayna EM, et al: Receptor tyrosine kinase signaling required for integrin avb5-directed cell motility but not adhesion to vitronectin. J Cell Biol 127:859-856, 1994 46. Kovalenko M, Gazit A, Bohmer A, et al: Selective plateletderived growth factor receptor kinase blockers reverse sis-transformation. Cancer Res 54:6106-6114, 1994 47. Novogrodsky A, Vanichkin A, Patya M, et al: Prevention of lipopolysaccharide-induced lethal toxicity by tyrosine kinase inhibitors. Science 264:1319-1322, 1994 48. Brown A, Jolly P, Wei H: Genistein modulates neuroblastoma cell proliferation and differentiation through induction of apoptosis and regulation of tyrosine kinase activity and N-myc expression. Carcinogenesis 19:991-997, 1998 49. Kanbe K, Chigira M, Watanabe H: Effects of protein kinase inhibitors on the cell motility stimulated by autocrine motility factor. Biochim Biophys Acta 1222:395-399, 1994 50. Businco L, Bruno G, Giampietro PG: Soy proteins for the
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prevention and treatment of children with cow’s milk allergy. Second International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, Brussels, 1996 (abstr) 51. Janas L, Ostrom KM: Tolerance of soy formulas with reduced phytate/phytoestrogens fed to healthy, term infants. Second International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, Brussels, 1996 (abstr) 52. Irvine CH, Shand N, Fitzpatrick MG, et al: Daily intake and urinary excretion of genistein and daidzein by infants fed soy- or
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Discussion J. Gosche (New Haven, CT): What happens to the cells? What is the effect on the cells? There is inhibition, but is it apoptosis, is it differentiation? S. Naraghi (response): Actually, it inhibits dedifferentiation. The clonogenic growth actually is the ability of one single tumor cell to build a cell colony, build a small tumor, and with R-TK inhibitors would inhibit this division and dedifferentiation, and it led to the apoptosis of the cells.
J. Gosche (New Haven, CT): Has anyone ever looked at the effect of these R-TK inhibitors on growth and development in immature animals? They may be causing more toxicity by affecting normal processes than which we see with chemotherapy. S. Naraghi (response): Not that I know of. But our next step would be to test the effect of R-TK inhibitors on animal studies. We know that it has inhibited growth and development in breast cancer. This was in in vitro studies.
Purpose: Children who undergo standard therapy for renal tumors are at an increased risk for treatment sequelae such as congestive heart failure, abnormal trunk development, and secondary malignancies. Therefore, research on the use of novel chemotherapeutic agents with fewer side effects is justified. Recent experimental evidence suggests that growth factor receptors such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) play an important role in growth and development of pediatric renal tumors especially that of Wilms’ tumor. In this study we investigated the effects of genistein, AG1478, and AG1295, from the class of growth factor receptor tyrosine kinase (GFR-TK) inhibitors, on proliferation and colonigenic growth of 2 pediatric renal tumor cell lines.
(P ⫽ .01 and 5E-06, 2-tailed t test, respectively) and that of SK-NEP-1 by 44% and 74% (P ⫽ .0001 and 9.9E-07). The mean percent growth inhibition at the above doses was 57% ⫾ 7.9% and 96% ⫾ 0.2% for G-401, and 47% ⫾ 11.2% and 60% ⫾ 2.7% for SK-NEP-1. AG1478 at concentrations of 1,000 and 5,000 mol/L inhibited the colonigenic growth of G401 by 75% and 78% (P ⫽ .0005 and 7.38E-06, respectively) and that of SKNEP-1 by 19% and 40% (P ⫽ .02 and .0001). The percent growth inhibition at the mentioned concentrations for G-401 were 53% ⫾ 9.3% and 63% ⫾ 6.3%, and for SK-NEP-1 were 55% ⫾ 14.5% and 65% ⫾ 20.1%, respectively. AG1295 did not appear to be as effective as AG1478.
Methods: The authors studied the effect of genistein (broadspectrum GFR-TK inhibitor), AG1478 (EGFR-specific GFR-TK inhibitor), and AG1295 (PDGFR-specific GFR-TK inhibitor) on proliferation and colonigenic growth of rhabdoid tumor of the kidney and Wilms’ tumor cell lines: G-401 and SK-NEP-1, respectively. The effect of genistein at concentrations of 0 to 200 µmol/L, and AG1478 and AG1295 at 0 to 10,000 mol/L were tested on proliferation by using a growth inhibition assay. Viable cell counts at each concentration were obtained by hemocytometer and trypan blue exclusion, and percent growth inhibition was calculated based on control cultures at the same time-point. As a measure of colonigenic survival, the percent inhibition of colony formation in drug-treated dishes was calculated based on the number of colonies (⬎50 cells) in control dishes.
Conclusions: This is the first experimental study on the use of GFR-TK inhibitors as a potential treatment for pediatric renal tumors. GFR-TK inhibitors such as genistein occur naturally in soybean foods and have been shown to reach therapeutic levels in blood after consuming a soybean-based diet. Considering the significance of growth factor receptor activity in Wilms’ tumor development, inhibition of GFR-TKs should be investigated as effective and potentially nontoxic adjunctive treatment for this childhood tumor. Furthermore, GFR-TK inhibitors may offer an effective alternative to the treatment of commonly fatal rhabdoid tumor of the kidney in children. J Pediatr Surg 35:884-890. Copyright 娀 2000 by W.B. Saunders Company.
Results: Genistein at concentrations of 25 and 50 µmol/L inhibited the colonigenic growth of G-401 by 37% and 79%
INDEX WORDS: Pediatric renal tumors, tyrosine kinase receptor inhibitors.
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this once uniformly lethal disease. Still, children who undergo standard therapy for Wilms’ tumor are at an increased risk for treatment sequelae such as congestive heart failure, abnormal trunk development, and secondary malignancies.1-3 In addition, 11% of the Wilms’ tumor patients present with an anaplastic variant of the disease that, despite advances in therapy, is still associated with high rates of recurrence and death.1 Rhabdoid tumor of the kidney is an uncommon renal tumor of childhood that is associated frequently with highly aggressive behavior, resistance to multiagent chemotherapy, and fatal metastasis.4 This, together with the late adverse effects of cancer treatment in children, justifies research on the future use of novel and perhaps minimally toxic chemotherapeutic agents. Increased expression of growth factor receptors (GFRs)
ILMS’ TUMOR is the most common primary malignant renal tumor in children, accounting for about 6% to 7% of all pediatric neoplasms. Advances in surgical techniques and postoperative care together with the sensitivity of the tumor to radiation and chemotherapy has led to a dramatic improvement in the prognosis of From the Department of Surgery, University of Vermont, Burlington, VT. Presented at the 1999 Annual Meeting of the Section on Surgery of the American Academy of Pediatrics, Washington, DC, October 8-10, 1999. Address reprint requests to Dennis W. Vane, MD, Department of Surgery, University of Vermont, Burlington, VT 05401. Copyright 娀 2000 by W.B. Saunders Company 0022-3468/00/3506-0015$03.00/0 doi:10.1053/js.2000.6907 884
Journal of Pediatric Surgery, Vol 35, No 6 (June), 2000: pp 884-890
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including epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and insulin-like growth factor receptor (IGFR) in human epithelial tumors is correlated with poor prognosis and short survival times in patients with carcinoma of the breast,5 lung,6 nasopharynx and mouth,7,8 bladder,9 esophagus and gastrointestinal tract,10-13 pancreas cancer,14,15 endometrium,16 and thyroid gland.17 In glioblastomas and sarcomas (bone and soft tissue) as nonepithelial tumors, growth factor receptor overactivity is linked to shorter survival and time to recurrence after treatment.18,19 Recent experimental evidence suggests that growth factor receptors such as EGFR and PDGFR play an important role in the growth and development of embryonic cancers such as osteosarcoma and Wilms’ tumors.20-22 It is the guiding hypothesis of this study that inhibiting the action of these receptors by suppressing their intrinsic tyrosine kinase activity can block the mitotic and colonigenic growth of pediatric renal tumors. To clarify this, we have used genistein and tyrophostins (AG1478 and AG1295) from the class of receptor tyrosine kinase (RTK) inhibitors.23 These agents have been shown to have no in vivo toxicity at effective therapeutic doses in a number of neoplastic and inflammatory diseases and potentially could be used as an alternative therapy for Wilms’ tumor or rhabdoid tumor of the kidney. MATERIALS AND METHODS For growth inhibition in this study 2 commercially available human renal tumor cell lines, G-401 and SK-NEP-1, from American Type Culture Collection (ATCC) were used. G401 line was derived from the rhabdoid tumor of the kidney of a 3-month-old boy and SK-NEP-1 line originates from cells of a malignant pleural effusion in a 25-year-old female patient with Wilms’ tumor.
Effect of Genistein, AG1478, and AG 1295 on Tumor Monolayer Cell Growth Cryopreserved G-401 and SK-NEP-1 cells were thawed rapidly and propagated in 75-cm tissue culture flasks (Falcon, Franklin Lakes, NJ) containing complete media. G-401 and SK-NEP-1 cells were grown in modified McCoy’s 5a media with L-glutamine supplemented with 10% and 15% heat-inactivated newborn calf serum, respectively, and 1% antibiotic-antimycotic solution (Gibco BRL, Grand Island, NY) under standard tissue culture conditions (100% humidity, 95% air, and 5% CO2). Subconfluent monolayers of cells were trypsinized and 1 ⫻ 105 were seeded into each well of a 12-well tissue culture plate (Falcon, Lincoln Park, NJ). A 0.1 mol/L stock solution of genistein (Calbiochem, La Jolla, CA) in DMSO (Sigma, St Louis, MO) was diluted in complete growth medium as described above to reach final concentrations of 10, 25, 50, 100 and 200 µmol/L. The concentration of DMSO in control media and at 200 µmol/L genistein concentration was 0.2%. Similarly, 0.01 mol/L stock solutions of tyrphostins AG1478 and AG1295 (Calbiochem, La Jolla, CA) in DMSO were diluted in complete growth media as previously described to reach concentrations of 100, 500, 1,000, 5,000, and 10,000 mol. The concentration of DMSO in control media and at 10,000 mol
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tyrphostin was 0.1%. Media containing each tested drug at abovementioned concentrations were added to each well 24 hours after plating of cells and were replenished every 24 hours with fresh complete medium containing the studied drug for 4 days. Cells were then harvested by trypsinization at 5 days, and viable cell counts were obtained by hemocytometer and trypan blue exclusion. Cell counts at each concentration were performed in triplicates. The percent growth inhibition was calculated based on control cultures using medium containing DMSO at the same time point.
Effect of Genistein, AG1478, and AG1295 on Colonigenic Growth of Tumor Cell Lines Cell suspensions of G-401 and SK-NEP-1 were incubated at the above doses of genistein, AG1478 and AG1295 separately for 24 hours. Next SK-NEP-1, and G-401 cells were centrifuged and washed in D-PBS, and seeded at a density of 1,000 cells in enriched complete medium (20% heat inactivated new born calf serum) in 60-mm culture dishes. A solid culture substrate was used here instead of semisolid agarose because these cells do not form colonies in agarose. G-401 cells form anchorage-dependent colonies and, SK-NEP-1 cells form nonadhering spheroids. The cultures were maintained under standard tissue culture conditions for at least 10 days. The number of colonies containing more than 50 cells in diameter were then counted at each drug concentration in each dish. The percent inhibition of colony formation (as the measure of inhibition of colonigenic survival) in drug-treated dishes was calculated based on the number of colonies (⬎50 cells in diameter) in control dishes.24
RESULTS
As explained earlier, effects of the receptor tyrosine kinase inhibitors genistein, AG1478 and AG1295 on renal tumor cell lines G-401 and SK-NEP-1 were measured in terms of inhibition of monolayer cell growth and colony formation on a solid substrate. These cells did not form colonies in 0.5% agarose, but formed spheroids and adhering colonies on a solid surface. Effect of Genistein on Tumor Monolayer Cell Growth and Colonigenic Growth At the doses of 25 and 50 µmol/L genistein appeared to suppress monolayer cell growth of tumor cells by 57% ⫾ 7.9% and 96% ⫾ 0.2% for G-401, and 47% ⫾ 11.2% and 60% ⫾ 2.7% for SK-NEP-1. Genistein inhibited colony formation of G-401 at doses of 25 and 50 µmol/L by 37% and 79% (P ⫽ .01 and 5E-06, respectively; 2-tailed t test) and that of SK-NEP-1 by 44% and 74% (P ⫽ .0001 and 9.9E-07, respectively; Figs 1, 2, 3A-C, 4A-C). Effect of AG1478 and AG1295 Tumor Monolayer Cell Growth and Colonigenic Growth AG1478 (EGFR-specific GFR-TK inhibitor) at concentrations of 1,000 and 5,000 mol/L inhibited the monolayer growth of tumor cells by 53% ⫾ 9.3% and 63% ⫾ 6.3% for G-401 and 55% ⫾ 14.5% and 65% ⫾ 20.1% for SK-NEP-1, respectively. AG1295 (PDGFR-specific GFR-TK inhibitor), however, did not appear to be as effective (Fig 5).
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Fig 1. Effect of genistein on monolayer cell growth of G-401 and SK-NEP-1 lines.
AG1478 at the above concentrations suppressed the colony formation of G-401 cells by 75% and 78% (P ⫽ .0005 and 7.38E-06, respectively; 2-tailed t test) and that of SK-NEP-1 by 19% and 40% (P ⫽ .02 and .0001). AG1295 at above concentrations did not significantly inhibit colony formation of G-401 cells: 9% and 7%, respectively (P ⫽ .41 and P ⫽ .66). AG1295 was similarly ineffective on SK-NEP-1 cells (Fig 6). DISCUSSION
Role of Growth Factor Receptor Tyrosine Kinases in Clinical Tumor Progression Activation of a growth factor receptor (GFR) such as EGFR, through the binding of its ligands (EGF or transforming growth factor [TGF-␣]), results in the induction of the receptor’s intrinsic tyrosine kinase (RTK) activity.25,26 This leads to autophosphorylation of intracellular tyrosine residues on the receptor, followed by tyrosine phosphorylation of various signaling proteins27 that lead to a series of cellular activities including proliferation and migration.28-30 As discussed earlier, increased expression of growth factor receptors including EGFR, PDGFR, and insulinlike growth factor receptor (IGFR) in human epithelial tumors is correlated with poor prognosis and short survival times in patients.5-17 In glioblastomas and sarco-
Fig 2. Effect of genistein on colony formation of G-401 and SK-NEP-1 cell lines.
Fig 3. SK-NEP-1 spheroids at 3 genistein concentrations. (A) 0 mol/L, (B) 25 mol/L, and (C) 50 mol/L. (Original magnification, ⴛ25.)
mas (bone and soft tissue) as nonepithelial tumors, growth factor receptor over activity is linked to shorter survival and time to recurrence after treatment.18,19 Intercepting the growth factor receptor activation cascade by using GFR-TK inhibitors has been, therefore, recognized as a target for anticancer drug design.23 Reports on the experimental in vitro and in vivo (animal experiments) use of GFR-TK inhibitors have shown encouraging results in blocking growth and invasiveness of a number of tumors such as breast cancer, colorectal carcinoma, glioblastoma multiforme, and leukemia.31-37
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Fig 5. Effect of AG1478 and AG1295 on monolayer cell growth of G-401 and SK-NEP-1 lines.
differentiation and growth of metanephric blastema has been described, the role of agents that control and regulate this process is not understood completely. It is, however, known that this process is coordinated by the timed and sequential interaction between the growth factors such as IGF, EGF, TGF-␣ and PDGF and their receptors. Therefore, loss of regulation of growth factor– mediated development and growth is believed to retard kidney differentiation and result in neoplastic transformation of metanephric blastema and formation of Wilms’ tumor.22,38 The origin of rhabdoid tumor of the kidney, however, is less certain. Previous findings have indicated that this tumor may arise from the metanephric blastoma as a result of a germline mutation. This suggestion is based on the observation that rhabdoid tumor commonly occurs together with primitive neuroectodermal tumor of the brain.39 So far the only growth factors implicated in the growth and development of rhabdoid tumor have been IGF I and II.40 The exact nature of their involvement remains unclear. Tumor Treatment Using Receptor Tyrosine Kinase Inhibitors
Fig 4. G-401 colonies at 3 genistein concentrations. (A) 0 mol/L, (B) 25 mol/L, and (C) 50 mol/L. Colonies were fixed using methyl alcohol and stained with methylene blue. (Original magnification, ⴛ40.)
Recent reports on the experimental in vivo use of selective RTK inhibitors have shown dramatic results in curing B-cell leukemia32 and preventing breast cancer.33,34 The most anticarcinogenic and antiproliferative group of these drugs, the isoflavones genistein and daidzein, occur naturally in soybeans and soy-protein
Growth Factor Receptors and The Biology of Wilms’ Tumor and Rhabdoid Tumor of Kidney Wilms’ tumor cells originate from the undifferentiated mesenchymal cells of metanephric blastema. Metanephric blastema, also called metanephrogenic mesenchyme, is that part of the mesoderm of the embryonic kidney that gives rise to the excretory tubules of the metanephros. Metanephros is the most caudally located part of the embryonic renal excretory organ, which will eventually serve as the permanent kidney in humans. Although the
Fig 6. Effect of AG1478 and AG1295 on colony formation of G-401 and SK-NEP-1 cell lines.
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foods41 and have been shown to reach effective levels in blood after consuming a soybean-based diet.42 Genistein is a potent and relatively specific inhibitor of receptor tyrosine kinase activity with a wide range of inhibiting concentration (IC)50 values (2.6 to 79 µmol/L) for proliferation of human tumor lines in vitro.34,43 Obtained through the diet, genistein can inhibit in vitro angiogenesis (endothelial cord formation inhibited at an IC50 of 5 µmol/L), and endothelial cell proliferation (IC50 of 150 µmol/L) stimulated by basic fibroblast growth factor (bFGF).44 Genistein inhibits plasminogen activator (PA) and PA-inhibitor-1 in vascular endothelium, which play a role in tumor angiogenesis.44 Genistein also inhibits the activity of topoisomerase II in vitro with IC50 values around 111 µmol/L.43 Previous experimental work has shown that genistein can block invasiveness of glioblastoma and breast cancer lines in organotypic coculture systems in vitro.31,35 Similar effects have been reported for synthetic RTK inhibitors such as tyrphostins as well.31,36,45 Tyrophostins are a group of synthetic simple organic compounds that specifically inhibit RTKs and not other protein kinases, by competing for the substrate binding site and not the ATP binding site (both are located on the intracellular domain), which is a common structural feature among all protein kinases. These compounds are, therefore, more potent, selective, and nontoxic inhibitors of RTKs than isoflavones (genistein and daidzein).23 For example, the tyrphostin AG1478 is a very potent and specific inhibitor of EGFR-TK activity (IC50 ⫽ 0.003 µmol/L) as compared with PDGFR-TK activity (IC50 ⬎ 100 µmol/L).46 The opposite relationship between effects exists for another member of this family; AG1295 (IC50 for PDGFR-TK inhibition is 0.5 µmol/L v little to no effect on EGFR-TK activity).46 Animal experiments using tyrphostins have shown that these compounds are nontoxic at up to 30 times their effective dose in vivo.47 In this study we examined the effect of the abovementioned RTK inhibitors on monolayer cell growth and colonigenic growth of 2 human renal tumor cell lines. Our results showed significant suppression of both proliferation and colony formation of the G-401 and SKNEP-1 lines by the EGFR selective RTK inhibitors genistein and AG1478 but not the PDGFR-specific AG1295. This suggests that perhaps EGFR signaling plays a role in the growth of these 2 tumor cell lines. Antitumor effects for RTK inhibitors have been shown in other childhood tumors such as neuroblastoma and osteosarcoma.48,49 Genistein suppresses the growth of neuroblastoma cell lines in vitro by inducing apoptosis and differentiation48 and blocks growth factor–induced migration of tumor cells in an osteosarcoma cell line.49 The significant effect of genistein on tumor cell growth in our study may be attributed to inhibition of RTK or
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topoisomerase II activity. Our preliminary results from mechanistic studies have so far shown that genistein at concentrations up to 50 µmol/L does not inhibit tumor cell growth by inducing apoptosis. As mentioned earlier, typical IC50 concentrations for topoisomerase activity as reported in the literature are greater than 100 µmol/L.43 Further experimental studies are still necessary to clarify the mechanism of action of these drugs. There have been to date no reports on any in vitro or in vivo use of RTK inhibitors as potential treatment for childhood renal tumors. The long-term goal of such experimental studies is to eventually proceed to animal experiments and human patient trials. In this connection the use of soybean isoflavones, such as genistein and daidzein, is particularly attractive because soy protein formulae have been widely used to feed infants with cow milk allergy since the early 1920s.50 A number of studies have shown that soy-based diets in full-term infants are well tolerated and have no effects on weight gain.51 Although bioavailability of soymilk isoflavones in children is still under investigation,52 a series of human trials has shown isoflavone (genistein and daidzein) plasma levels of up to 4 µmol/L after oral doses of 2 mg/kg body weight as a part of soymilk formulations in adults.42 Plasma isoflavone concentrations in 4-month-old infants fed soy-based formula have been reported to be between 3 and 6 µmol/L.53 Such low plasma concentrations may not justify the use of oral isoflavones in its current form as single-agent chemotherapy. There is, however, emerging evidence that even at a low dose there may be a significant synergistic antiproliferative effect with standard chemotherapy for some tumors.54,55 The significance of this for pediatric tumors such as Wilms’ tumor may be a reduction in the current dose recommendations for chemotherapy and radiation therapy without compromising their effectiveness but perhaps reducing the devastating treatment sequelae. Furthermore, isoflavones may offer a new therapeutic alternative where present multidrug chemotherapy has achieved little in treatment of rhabdoid tumor of the kidney in children. REFERENCES 1. Green DM, D’Angio GJ, Beckwith JB, et al: Wilms tumor. CA Cancer J Clin 46:46-63, 1996 2. DeLaat CA, Lampkin BC: Long-term survivors of childhood cancer: Evaluation and identification of sequelae of treatment. CA Cancer J Clin 42:263-282, 1992 3. Meadows AT, Baum E, Fossati-Bellani F, et al: Second malignant neoplasms in children: An update from the Late Effects Study Group. J Clin Oncol 3:532-538, 1985 4. Weeks DA, Beckwith J, Mierau GW, et al: Rhabdoid tumor of kidney. Am J Surg Pathol 13:439-458, 1989 5. Rudland PS, Fernig DG, Smith JA: Growth factors and their receptors in neoplastic mammary glands. Biomed Pharmacother 49:389399, 1995 6. Occleston NL, Walker C: Production of multiple growth factors
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Discussion J. Gosche (New Haven, CT): What happens to the cells? What is the effect on the cells? There is inhibition, but is it apoptosis, is it differentiation? S. Naraghi (response): Actually, it inhibits dedifferentiation. The clonogenic growth actually is the ability of one single tumor cell to build a cell colony, build a small tumor, and with R-TK inhibitors would inhibit this division and dedifferentiation, and it led to the apoptosis of the cells.
J. Gosche (New Haven, CT): Has anyone ever looked at the effect of these R-TK inhibitors on growth and development in immature animals? They may be causing more toxicity by affecting normal processes than which we see with chemotherapy. S. Naraghi (response): Not that I know of. But our next step would be to test the effect of R-TK inhibitors on animal studies. We know that it has inhibited growth and development in breast cancer. This was in in vitro studies.