• Introduction
• Antibodies
• Cell culture
• Cell viability assay
• FAK antisense oligonucleotide treatment
• Western blot analysis
• Statistical Analysis
• Results
• The cytotoxic effect of genistein on G361 cell viability
• Effect of genistein on 125 kDa tyrosine phosphoryla-tion of G361 and HaCaT cells
• Effect of genistein on FAK expression in G361 and HaCaT cells
• Effect of FAK antisense oligonucleotide on the viability of melanoma cells
• The regulation of cell cycle related proteins
• Discussion
• Acknowledgement

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Introduction

 Melanoma cells require adhesion through integrin recep-tors for their survival and growth. Attachment to the extra-cellular matrix suppresses apoptosis in these cells [1]. Clus-ters of integrins, occurring in focal adhesion contact sites,  interact with the matrix during cellular attachment. Focal ad-hesion is not only important for attachment, but also essen-tial to subsequent cell spreading and motility [2]. Integrin-matrix interactions regulate cell growth and apoptosis by ini-tiating signal transduction pathways [3]. FAK is a major sig-naling mediator, the activation of which requires both inte-grin attachment and cell spreading [4].

 It has been shown that Focal adhesion kinase (FAK) is overexpressed in breast, colon, and thyroid cancers [5-7], whereas normal tissues express little detectable FAK. The overexpression of FAK in tumors is likely to affect three functions as follows: motility, adhesion, and survival. FAK is thought to play a role in adhesion-mediated survival beca-use overexpression of a constitutively activated form of FAK in Madin-Darby canine kidney cells has been shown to confer resistance to apoptosis following loss of adherence [8]. FAK overexpression in Chinese hamster ovary (CHO) cells cau-sed an increase in migration [9], suggesting that FAK may play a role in motility of CHO cells. Although these exper-iments were performed in normal cells, they raise the possi-bility that tumor cells upregulate FAK expression in order to leave their tissue of origin, invade their surrounding stroma, and migrate into new environments. However, FAK overex-pression has also been demonstrated in preinvasive tumors [10], suggesting a role for FAK in tumors that occurs before development of anchorage-independent growth potential.

 Low rates of breast, colon, and prostate cancers have been reported in Asian countries, such as China and Japan, rela-tive to the United States [11]. Epidemiological studies have suggested that a diet rich in isoflavonoids may play an im-portant role in cancer prevention [12]. Genistein is an isof-lavone [13], believed to be a metabolite of soy produced by the gut floral bacteria [14]. Genistein has a heterocyclic, dip-henolic structure similar to estrogen [15] and was shown to be a potent inhibitor of epidermal growth factor receptor tyro-sine kinase [16]. The protein products of approximately one- half of the known oncogenes have been shown to be mem-brane-bound receptors with tyrosine kinase activity or intra-cellular proteins undergoing or catalyzing tyrosine phosp-horylation [17]. The anticancer effects of soy products could be attributed to genistein [18] perhaps by modulating tyrosine kinase activity. Genistein has also been shown to be an inhibitor of angiogenesis in vitro [19], which plays an important role in tumor growth and metastasis. It also inhibits DNA to-poisomerases I and II in vitro [20-21] and ribosomal S6 kinase [22] and induces differentiation of cancer cell lines [23]. Genis-tein was shown to induce cell cycle progression arrest at the G2/M phase and induced apoptosis in breast, gastric and pros-tate cancer cell lines [24-26]. Therefore, this study was under-taken to investigate the effect of genistein on the expression of FAK and cell cycle related proteins in G361 melanoma cell line, comparing with normal HaCaT keratinocytes. Amersham international (Buckinghamshire, UK).

Antibodies

 Monoclonal antibodies (MoAb) mouse anti-human FAK (05-1139) and PY20 (05-947) were purchased from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal anti-human p53 (SC-99), p21 (SC-53393), cdc2 (SC-54) and cyc-lin B1(SC-7393), and 14-3-3γ (SC-25276) were from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase labelled anti-mouse IgG were purchased from Amersham Bioscien-ces (UK).

Cell culture

 The G361 human melanoma cell line was purchased from ATCC (Rockville, USA). Cells were maintained at 37℃ with 5% CO₂  in air atmosphere in minimum essential medium (Eagle) with 2 mM L-glutamine and Earle’s BSS adjusted to contain 1.5 μg/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate, and supplemen-ted with 10% FBS. Cells were maintained in Dulbecco’s modi-fied Eagle’s medium with 10% FBS.

Cell viability assay

 The viability of cultured cells was estimated by MTT assay. In the MTT assay, cells were placed in a 96-well plate and incubated for 24 h. Then cells were treated with various concentrations of genistein for 24 h. And then, the cells were treated with 1 mg/ml of MTT in growth medium. Cells were incubated at 37℃, 5% CO₂ for 4 h. The medium was aspirated and the formazan crystals, which are formed from MTT by NADH-generating dehydrogenases in metabolically active cell, were dissolved in 200 ㎕ DMSO. Cell viability was evaluated in comparison to the control culture (taken as 100%) by measuring the intensity of the blue color (OD at 570 nm) by a multi-well reader (Quant, Bio-Tek, Highland Park, USA). The assay was performed in triplicate.

FAK antisense oligonucleotide treatment

 The FAK antisense oligonucleotide was 5'-TTT-CAA-CCA- GAT-GGT-CAT-TC-3'. Oligonucleotides were added at 300 nM, with 3μl of lipofectin (Invitrogen, Life Technologies, Inc., Carlsbad, California, USA) per ml of OptiMEM I me-dium (Life Technologies, Inc.) per 100 nM oligonucleotide, and incubated for 4 h. Subsequently, the cells were washed and complete medium was added for 20 h. The control sam-ple was incubated for 4 h in OptiMEM I and lipofectin, but no oligonucleotides.

Western blot analysis

 For protein analysis, cells were lysed with RIPA buffer (10 mM Tris/HCl, pH 7.2, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 158 mM NaCl, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride) on ice for 1 h. Lysate were clarified by centrifugation at 12,000 revolution per min for 20 min at 4℃, and then the supernatant was obtained. The protein contents of the lysate were determined using the Bio-Rad Protein Assay (Bio-Rad laboratoris Hercules, CA). The 20 μg protein was mixed with equal volume of elect-rophoresis buffer (10 mM Tris/HCl, 200 mM DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol). After heating, the protein was resolved on polyacrylamide SDS gels and transferred to nitrocellulose membrane. After transfer, the membranes were blocked with blocking reagent (5% non-fat milk in distilled water) for 1 h and then the membranes were incubated with primary antibody. The membranes were incubated for 1 h with the corresponding secondary anti-body, diluted in the above blocking reagent. After three final washes, the membranes were treated with chemilumi-nescence reagent. All the procedures were done at room temperature.

Statistical Analysis

 Three independent experiments were performed triplica-tes. The results of treated and control groups were compared for statistical significance (p<0.05) using paired T-test statisti-cal method by SPSS (SPSS version 18 for windows, SPSS Inc, USA).

Results

The cytotoxic effect of genistein on G361 cell viability

 Genistein preferentially had a significant time-dependent inhibitory effect on the viability of G361 cells compared with HaCaT. (Fig. 1). The cytotoxic effect of 50 µM genistein sig-nificantly decreased the proliferation of G361 cells, the via-bility of HaCaT cells was slightly affected by genistein treat-ment in the course of time.

Fig. 1. Preferential cytotoxic effects of genistein treatment on the viability of G361 melanoma cells. Cells were treated with 50 µM genistein for indicated time periods and cell numbers were mea-sured by MTT assay (G361, 24-72 h p<0.05). Four indepen-dent assays were performed and data shown are the mean ±SD of the means obtained from triplicates of each assay.

Effect of genistein on 125 kDa tyrosine phosphoryla-tion of G361 and HaCaT cells

 In order to demonstrate that G361 cells are much more sensitive to genistein than normal HaCaT cells, we mea-sured the expression of 125 kDa phosphotyrosine kinase in both cells after genistein treatment (Fig. 2). Because genis-tein is known to be a inhibitor of protein tyrosine kinase. In the case of G361 cells, a small quantity of phosphotyrosine kinase was expressed and it was almost disappeared at 16 h and 24 h by genistein. Relatively to G361 cells, HaCaT cells expressed more quantity of 125 kDa phosphotyrosine kina-ses, which were slightly decreased by genistein treatment.

Fig. 2. Inhibition of 125 kDa tyrosine phosphorylation by the genistein. After two cell lines were treated with 50 µM genistein for 16 and 24 h, cells were lysed and separated on SDS-PAGE, transferred to nitrocellulose membrane and reacted with anti-phosphotyrosine antibody.

Effect of genistein on FAK expression in G361 and HaCaT cells

 FAK is the most representative protein among 125 kDa phosphotyrosine kinase proteins. Thus, we investigated the expression of FAK in two cells treated with genistein (Fig. 3). Genistein treatment resulted in the decreased FAK protein levels found on Western blots performed. The expression le-vel of FAK of G361 cells was higher than in HaCaT cells, dis-like the expression pattern of 125 kDa phosphotyrosine kinases.

Fig. 3. Inhibition of the expression of FAK protein by the geni-stein. After two cell lines were treated with 50 µM genistein for 16 and 24 h, cells were lysed and separated on SDS-PAGE, trans-ferred to nitrocellulose membrane and reacted with anti-FAK antibody.

Effect of FAK antisense oligonucleotide on the viability of melanoma cells

 In order to examine that FAK takes the responsibility for different sensibility of these cells to genistein, FAK antisen-se oligonucleotide was treated to both cells. Treatment of FAK antisense oligonucleotide significantly decreased the via-bility of G361 cells compared with HaCaT, which were rarely affected by FAK antisense oligonucleotide (Fig. 4).

Fig. 4. Effect of FAK antisense oligonucleotide on the viability of G361 and HaCaT cells. After two cell lines incubated with FAK antisense oligonucleotid for 24 h, cell numbers were mea-sured by MTT assay (G361, 24 h; p<0.05). Four independent assays were performed and data shown are the mean ±SD of the means obtained from triplicates of each assay.

The regulation of cell cycle related proteins

 Next we investigated the modulation of cell cycle regula tory proteins in G361 cells by genistein treatment. The exp-ression of p53 was not observed at indicated time after geni-stein treatment. While the expression of p21 was increased up to 24 h, the down-regulation of cdc2 and cycline B was detected in time-dependent manner (Fig. 5). When cell cycle arrest at specific phase was examined, genistein induced the cell cycle arrest at G₂/M phase (Fig. 6).

Fig. 5. Western blot analysis of cell cycle regulation proteins in G361 melanoma cells treated with 50 µM genistein. The exp-ression of p21 were increased up to 24 h, whereas the expres-sion level of cdc2 and cyclin B were decreased time‐depen-dently. The expression of p53 was not observed.

Fig. 6. The kinetic analysis of the effect of 50 µM genistein on G361 cell cycle progression. The increase in the G2/M phase cell percentage was shown.

Discussion

 Resistance to induction of apoptosis may be a mechanism by which tumor cells survive during anti-neoplastic treat-ments [27]. Melanoma is notoriously resistant to anti-neop-lastic therapy, with only one Food and Drug Administration (FDA)-approved chemotherapeutic agent (dacarbazine), and no better than 25% response rates for any given agent [28].

 Previous studies have demonstrated FAK overexpression in melanoma cell lines [29-30]. Recently, using immuno-histochemical staining, Smith  et al. (2005) reported that FAK was overexpressed in 89 samples of primary human mela-noma, and the level of expression on all of the tumors was greater than the level of expression in melanocytes in the adjacent normal epidermis [31]. Given its probable role in mediating resistance to apoptosis, they sought to determine whether the downregulation of FAK in melanoma cells would confer a greater sensitivity to anti-neoplastic agents. FAK is required for cell survival in adhesion-dependent cells, and autophosphorylation of a major  tyrosine site is needed to perform this function [32]. Cell attachment induces FAK auto-phosphorylation on Tyr-397, and this allows the SH2 dom-ains of the Src family and other kinases to bind. Src then phosphorylates at least five Tyr residues in FAK. Phosphor-lyation of some of these residues leads to the activation of the mitogen-activated protein kinase cascade [33]. Therefore, FAK is thought to have various functions, ranging from the regulation of focal adhesion turnover to the prevention of apop-tosis [34]. Moreover, inhibition of FAK expression causes apoptosis in several human tumor cell lines including mela-noma [35].

 Genistein is a known inhibitor of protein-tyrosine kinase (PTK), which may attenuate the growth of cancer cells by inhi-biting PTK mediated signaling pathway [36-37]. PTKs are known to play key roles in carcinogenesis, cell growth and apoptosis [38]. It has been reported that genistein is a potent inhibitor of cell proliferation, oncogenesis and clonogenic ability of animal and human cells [39]. Experiments have shown that genistein inhibits growth of cancer cells including leuke-mia, lymphoma, neuroblastoma, gastric, breast and prostate cancer cells [24-26,40] .

 This study demonstrates that genistein has a preferential cytotoxic effect on G361 melanoma cells compared with HaCaT normal keratinocytes. Genistein decreased not only the exp-ression of 125 kDa phosphotyrosine kinase but also FAK in G361 melanoma cells. We also observed the alteration of cell cycle related proteins. It has been reported in the literature that genistein can cause G₂/M arrest in many other tumor cells [41-43]. Cells progressing through the cell cycle are control-led by the activation of a special family of protein kinases called the cyclin-dependent kinases (CDKs) [44]. For exam-ple, at the conclusion of the G₂ phase, CDKs phosphorylate and activate a set of proteins that function to promote mito-sis and cytokinesis. The G₂/M phase transition is controlled by the cyclin B-cdc2 complex, which is regulated by phosp-horylation [45]. The 15-tyrosine and the 161-threonine resi-dues of cdc2 must be phosphorylated first and, subsequent-ly, the 15-tyrosine residue must be dephosphorylated to ac-tivate the cyclin B-cdc2 complex activity. It is possible that genistein, a PTK inhibitor, may inhibit the activity of the PTK and, in turn, may deactivate the cyclin B-cdc2. It may, therefore, disturb the whole process of this phosphorylation- dephosphorylation chain reaction of tyrosine residues of cdc2 kinase at the very beginning, leading finally to G₂/M arrest. Although p21, a CDK inhibitor, has been reported to induce G₁ arrest [46], the accumulated evidence has shown that upre-gulation of p21 expression may also be associated with G₂/M phase arrest in the cell cycle [42-43]; the latter is consistent with our results. In this study, the p21 level in the genis-tein-treated G361 cells increased significantly. Taken toge-ther, genistein inhibits the proliferation of G361 melanoma cells via suppressing FAK expression and regulating cell cycle gene. These results suggest that genistein may be a good st-rategy for melanoma treatment.

Acknowledgement

 This work was supported by a 2-Year Research Grant of Pusan National University.