J Pharmacol Sci 103, 247 – 251 (2007) Journal of Pharmacological Sciences ©2007 The Japanese Pharmacological Society Short Communication Dietary Iberin Inhibits Growth and Induces Apoptosis in Human Glioblastoma Cells

Unmesh Jadhav1, Ravesanker Ezhilarasan1, Steven F. Vaughn2, Mark A. Berhow2, and Sanjeeva Mohanam1,* 1Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, One Illini Drive Peoria, IL 61605, USA 2New Crops and Processing Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service-USDA, 1815 North University St., Peoria, IL 61604, USA

Received August 10, 2006; Accepted November 11, 2006

Abstract. In this study, we evaluated the antiproliferative and proapoptotic effects of the isothiocyanate iberin, a bioactive agent in species, in human glioblastoma cells. The human glioblastoma cell cultures were treated with different concentrations of iberin and tested for growth inhibition, cytotoxicity, induction of apoptosis, and activation of caspases. Iberin inhibited growth of tumor cells in cell proliferation assays, enhanced cytotoxicity, and induced apoptosis by activation of caspase-3 and caspase-9. Findings from this study could provide a basis for potential usefulness of the diet-derived isothiocyanate iberin as a promising therapeutic micronutrient in the prevention/intervention of brain tumors.

Keywords: iberin, glioblastoma, apoptosis

Isothiocyanates, plant-derived dietary compounds, are activities both in vitro and in vivo (2, 4 – 7). There are promising chemopreventive agents since they are few studies on the isothiocyanate iberin ( generally non-toxic substances that interfere with the sulfoxide analog) (Fig. 1A) in comparison to sulfora- process of cancer development or carcinogenesis. phane and phenylethyl isothiocyanate, and its antitumor Several natural and synthetic have properties are not thoroughly examined. In the present demonstrated cancer-preventive properties in animals study, we investigated the effect of the isothiocyanate treated with chemical carcinogens, including polycyclic iberin on the growth of human glioblastoma SNB19 aromatic hydrocarbons and nitrosamines (1 – 3). cells. Our findings indicate that isothiocyanate-mediated Tumorigenesis is a multistage process with an accumu- apoptosis in glioblastoma cells is associated with acti- lation of genetic alterations. Suppression of cell vation of caspases. proliferation and induction of differentiation and Iberin was isolated from Lesquerella fendleri apoptosis during the promotion and progression stages seedmeal as described previously (8). The human are ideal preventive devices. Glioblastoma is the most glioblastoma SNB19 cells were cultured in DMEM common malignant tumor of the adult central nervous supplemented with 10% fetal bovine serum, penicillin system and is hallmarked by high proliferation of tumor (100 units/mL), and streptomycin (100 µg/ml) and cells with increased cellularity and necrosis. Failure to maintained at 37°C in a 95% air / 5% CO2 humidified respond to standard therapy including surgery, radiation, incubator (9). Stock solution of iberin was prepared in and chemotherapy prompts the development of DMSO, and an equal volume of DMSO (final concentra- alterative treatment modalities. Recent studies indicate tion was less than 0.1%) was added to controls. that natural isothiocyanates such as sulforaphane and Glioblastoma cells were treated with iberin at the possess strong antitumor indicated concentrations or the equivalent volume of vehicle (DMSO). The antiproliferative effects of iberin *Corresponding author. [email protected] were investigated by the widely used MTT cell prolifer- Published online in J-STAGE ation assay. To assess cell cytotoxicity, lactate dehydro- doi: 10.1254/jphs.SC0060148

247 248 U Jadhav et al genase (LDH) leakage was determined in the extracellu- lar cell-culture medium using the CytotoTox 96 Non- Radioactive Cytotoxicity Assay Kit (LDH) from Promega (Madison, WI, USA) according to the manu- facturer’s recommended protocol. Cells were treated with different concentrations of iberin for 24 h and were stained with FLICA (Immuno- chemistry Technologies Bloomington, MN, USA) fol- lowing the manufacturer’s instructions. Subsequently the cells were stained with 4',6-diamino-2-phenylindole (DAPI) to detect the apoptosis induced activation of nuclear fragmentation in situ and examined by fluores- cence microscopy. For Western blot analysis, cells were extracted in a buffer solution containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin on ice for 20 min. A 25-µg protein sample was analyzed by SDS-PAGE, and caspase-3 and caspase-9 protein expression levels were determined by immuno- blotting with the following monoclonal antibodies: Caspase-3 (Cell Signaling Technology, Beverly, MA, USA) and Caspase-9 (Cell Signaling Technology). For detection, the ECL detection system (Amersham Biosciences, Buckinghamshire, England) was used according to the manufacturer’s protocol. Equal loading of gels was confirmed by reincubation of the membranes with monoclonal antibody of β-actin (Abcam, Fig. 1. Morphological changes, growth inhibition, and cytotoxicity in human glioblastoma SNB19 cells after treatment with iberin. Cambridge, MA, USA). Statistical significance of the A: Chemical structure of iberin. B: Morphological changes to SNB19 experimental results was determined by Student’s t-test. cells after exposure to vehicle or 25 µM iberin for 24 h. Representa- For all analyses P<0.05 was accepted as a significant tive photos are shown. Scale bar = 50 µm. C: Cells were plated and probability level. allowed to grow for 24 h. The cells were treated with variable concentrations of iberin for 24 h and the viability was measured by The morphological changes by light microscopy in the metabolic-dye based MTT assay. Results are expressed as the iberin-treated cells are shown in Fig. 1B. We observed mean ± S.D. of 4 independent experiments done in duplicate. cell contraction and nuclear condensation in glioblas- *P<0.05, **P<0.01 vs respective control. D: Cells were exposed to toma cells treated with iberin. As shown in Fig. 1C, varying concentrations of iberin for 24 h, and LDH release into the medium was measured as described in the Materials and Methods iberin had a marked dose-dependent effect on SNB19 section. The data plotted are from 4 experiments done in duplicate cell growth. The growth was inhibited by 23%, 38%, and values are each a mean ± S.D. *P<0.05, **P<0.01 vs vehicle and 50% in cells exposed to 1.0, 2.5, and 25 µM iberin, treatment alone. respectively, as compared to the vehicle (DMSO)- treated control cells. An increase in the number of plasma-membrane-damaged cells results in an increase treatment with 1 µM iberin, cells clearly exhibited in LDH activity in the culture supernatant. Figure 1D significant morphological changes and chromosomal shows the concentration-dependent LDH release from condensation, which is indicative of apoptotic cell death iberin-exposed tumor cells. As shown in Fig. 1D, (Fig. 2A). Such results imply that the cytotoxic action of concentrations as low as 1.0 µM iberin caused a signifi- iberin was due to its ability to induce apoptosis. The cant increase in LDH release over the DMSO control detection of activated caspases by application of FLICA group (P<0.05), indicating that iberin exposure causes was performed with fluorescence microscopy. In the significant damage to the plasma membrane of the tumor present study a strong correlation was also seen between cells. the percentage of cells labeled with FLICA and those To know whether inhibition of growth by iberin cells labeled with DAPI exhibiting nuclear fragmenta- treatment is involved in induction of apoptosis in tion (Fig. 2A). These findings demonstrate that iberin SNB19 cells, we examined the nuclear morphology of activates induction of apoptosis in human glioblastoma cells with the fluorescent dye DAPI. Within 24 h of cells (Fig. 2B). Iberin Induces Apoptosis in Glioblastoma 249

Fig. 2. Effects of iberin treatment on induction of apoptosis in human SNB19 glioblastoma cells. The human glioblastoma SNB19 cells were exposed to different concentrations of iberin for 24 h. An equal volume of vehicle was added to the controls. To detect activation of caspases and chromatin condensation and fragmentation, cells were stained with DAPI and FLICA, respectively, and subsequently apoptotic cells were quantified by fluorescent microscopy. A: Representative fluorescence microscopy of DAPI and FLICA staining at the iberin concentration of 25 µM iberin. Scale bar = 50 µm. B: Percentage of apoptotic cells quantified by FLICA staining are shown and each column represents the mean ± S.D. of the data obtained from four to six independent experiments. *P<0.05, **P<0.01 vs vehicle treatment alone.

Fig. 3. Effects of iberin treatment on caspases in human glioblastoma cells. A: Cells were incubated with different concentrations of iberin for 24 h, lysed, and cellular proteins were separated by 12% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with the anticaspase-3 and -9 antibodies. Proteins were visualized by the ECL detection system. Actin was used as an internal control. The levels of cleaved caspase-3 (B) and procaspase-9 (C) were quantitated by densitometry, normalized to actin, and expressed as a percentage of the vehicle-treated value, which was set to 100%. The values are the mean ± S.D. of three different experiments. *P<0.05, **P<0.01 vs vehicle treatment alone.

We evaluated caspase-3 by measuring relative levels trated in Fig. 3, A and C, iberin caused a down- of activated caspase-3 by Western blotting. As shown regulation of the procaspase-9 form in SNB19 cells. in Fig. 3, A and B, the immunoblotting data indicated an These data point towards an involvement of the increase in cleaved caspase-3, demonstrating the mitochondria-mediated pathway in apoptosis induction participation of caspase-3 in iberin-induced apoptosis. by iberin in human glioblastoma SNB19 cells. We also examined the activation of caspase-9 in order to Glioblastoma is the most common malignant tumor examine the involvement of the mitochondrial pathway of the adult central nervous system and relapses with in apoptosis induction in iberin-treated cells. As illus- high rates of mortality despite multimodal therapies. 250 U Jadhav et al

Different epidemiological studies have indicated that In conclusion, we have demonstrated that the diet and cancers are closely associated and people who cruciferous vegetable derivative iberin is consume higher amount of fruits and vegetables have a able to inhibit growth of SNB19 cells by inducing lower risk of various types of cancers (10, 11). Several apoptosis at low concentrations, and the induction of studies demonstrate that and their main apoptosis is associated with the mitochondria-mediated breakdown products, for example, isothiocyanates and apoptotic pathway. Therefore, we speculate that the , which are present in , induction of apoptosis observed in this study may exhibit protective activities against cancers (2, 12). provide a distinct mechanism for the cancer therapeutic Isothiocyanates are known to inhibit the growth of and chemopreventive function of iberin, and further cancer cells, but the mechanisms are still only partially studies aimed at the determination of the therapeutic understood. The isothiocyanate iberin has been reported effects of iberin on glioblastoma growth in vivo should to exhibit certain biological effects (5, 13, 14), but its provide interesting insights on the potential clinical anticancer mechanism is still elusive. Consequently, the usefulness of this dietary constituent in the treatment present study was undertaken to shed more light on the of brain tumors. mechanisms by which iberin exerts its anti-proliferative properties in cultured human glioblastoma cells. 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