Proteome Analysis Of Neurite Differentiation Of Phytoestrogentreated Pc12 Cells Preliminary results

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Hiroko Isoda, Mariko Seki, Terence P. N. Talorete, Junkyu Han

Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan

Abstract: A novel isoflavone, 7, 2', 4'-trihydroxyisoflavone-4'-O-P- D-glucopyranoside was isolated from the aerial part of Crotalaria sessiliflora. The isoflavone glucoside was found to enhance the proliferation of the MCF-7 human breast cancer cell line, which possesses estrogen receptors (ERs) and responds in culture to estrogen. The estrogenic property of the isoflavone glucoside was blocked by the known ER antagonist tamoxifen, indicating the involvement of the ER. Furthermore, the isoflavone was found to enhance the acetylcholinesterase (AChE) activity of the rat neuronal cell line PC12 treated with a low concentration (0.1 ^M) of nerve growth factor (NGF). In this study, it was shown that the phytoestrogen enhances AChE activity in PC 12 cells by binding to the ER; the actual mechanism will be determined by proteome analysis.

Key words: Phytoestrogens, PC12, acetylcholinesterase

1. Introduction

Estrogenic compounds isolated from plants are commonly known as phytoestrogens and are known to exert a positive impact on human health (Knight and Eden 1996; Lamartiniere et al. 1995; Deodato et al. 1999). Although their chemical structures are relatively different from each other, they show some structural similarity: all contain a pharmacophore similar to that found in estradiol and diethylstilbestrol (Tham et al., 1998). Isoflavones are typical phytoestrogens that are found mostly in leguminous plants, particularly soybeans. Two major isoflavones found in soybeans are daidzein and genistein.

In the course of our study to find new antioxidative compounds from plants, we isolated a new isoflavone glucoside, 7,2',4'-tri-hydroxy isoflavone-4'-0-P-D-glucopyranoside from a BuOH soluble fraction obtained from the aerial part of C. sessiliflora (Leguminosae). Although this compound did not show antioxidative activity, its structure

resembled that of the phytoestrogen daidzein. Thus, we investigated the estrogenic activity of the compound by E-screen assay using the human breast cancer cell line MCF-7. This method is widely used as a rapid and straightforward assay to determine estrogenic activity of chemicals (Soto et al. 1995).

In a previous study, Isoda et al. (2002) reported that genistein and daidzein enhance the acetylcholinesterase (AChE) activity of the rat neuronal cell line PC12 at concentrations as low as 0.08 ^M by binding to the estrogen receptor (ER). Results have shown that this enhancement was effectively blocked by the known estrogen receptor antagonist tamoxifen, indicating the involvement of the ER in AChE induction. That genistein and daidzein are estrogenic were confirmed in a cell proliferation assay using the human breast cancer cell line MCF7. This proliferation was also blocked by tamoxifen, again indicating the involvement of the ER. On the other hand, incubating the PC12 cells in increasing concentrations of 17 B-estradiol (E2) did not lead to enhanced AChE activity, even in the presence of genistein or daidzein. This suggests that mere binding of an estrogenic compound to the ER does not necessarily lead to enhanced AChE activity. Moreover, the effect of the phytoestrogens on AChE activity cannot be expressed in the presence of E2 since they either could not compete with the natural ligand in binding to the ER or that E2 downregulates its own receptor. The study clearly suggests that genistein and daidzein enhance AChE activity in PC 12 cells by binding to the ER; however, the actual mechanism of enhancement is not known.

In this study, we also determined if the estrogenic activity of the isolated isoflavone was effectively blocked by the known ER antagonist tamoxifen to determine if the isoflavone glucoside can bind to the classical estrogen response element (ERE) and mediate gene transcription. We also provide preliminary results of an ongoing study of proteins from PC12 cells treated with daidzein using two-dimensional (2D) electrophoresis. Mass spectrometry of the protein spots of interest follows next. In 2D electrophoresis, the first dimension separates proteins according to their isoelectric point, while the second dimension separates them according to their molecular weight.

2. Materials and methods

2.1. Cell lines and maintenance

The cell lines used in this study and their maintenance were previously described (Isoda et al., 2002).

2.2 Acetylcholinesterase and cell proliferation assays

The acetylcholinesterase and cell proliferation assays were previously described (Isoda et al., 2002).

2.3. Proteome analysis

The cells were washed twice with tris-buffered sorbitol (10 mM Tris, 25 mM sorbitol pH 7), after which, one volume of the buffer was added, the cells then scraped and transferred to a polyallomer micro ultracentrifuge tube. Four volumes of lysis/extraction solution (7M urea, 2M thiourea, 4% CHAPS, 25 mM spermine base, 1mM EDTA, 50mM DTT, 4mM AEBSF) was immediately added and mixed by placing a piece of parafilm over the tube and inverting it several times. Extraction was carried out at room temperature for 60 min with occasional mixing. The sample was then centrifuged at 130,000 g for 1 h at 15 C. The protein-containing supernatant was kept and the protein concentration was determined using the 2D Quant Kit (Amersham). For first dimension, immobilized pH gradient (IPG) strips (Amersham) were rehydrated overnight with 350 ^g of protein (in-gel rehydration) according to the manufacturer's instructions. Using a Multiphor II apparatus, the proteins were then separated according to their isoelectric point under conditions provided in the 2D protocol (Amersham) with some modifications. For second dimension, the isoelectrically focused IPG strips were then equilibrated with DTT and iodoacetamide following the Amersham protocol. Each strip was then applied onto a ready-made SDS 12-14 Excel Gel® (Amersham) and the proteins separated according to their molecular weight using SDS-PAGE. Immediately after electrophoresis, the gels were fixed overnight in 30% ethanol and 0.5% glacial acetic acid. The gels were then stained with Coomassie brilliant blue (CBB) using PhastGel™ BlueR (Amersham). The stained gels were scanned using ImageScanner™ (Amersham) and the spots analyzed using the ImageMaster™ 2D software (Amersham).

3. Results and Discussion

Estrogen activity

Phytoestrogens or hormonally active agents can recognize the estrogen receptor and trigger cell proliferation in estrogen-responsive cells (Soto et al. 1991; White et al. 1994). Such compounds can also antagonize the effect of natural hormones, react directly or indirectly with the receptor, alter the pattern of synthesis of natural hormones, and even alter the hormone receptor level (Soto et al. 1995).

The estrogenic activities of isolated compounds were investigated by E-screen assay using the MCF-7 human breast cancer cell line. Daidzein and genistein, the best known naturally occurring nonsteroidal estrogens, were used as positive controls. Cell proliferation was measured by a colorimetric MTT assay to estimate the number of cells as the end point (Mossman 1983).

Results in Figure 1 show that when the MCF-7 cells were incubated for 6 days in the presence of 7,2',4'-trihydroxy isoflavone-4'-0-ß-D-glucopyranoside, there was a significant increase in cell proliferation compared to control (P< 0.01, t test). The proliferation of MCF-7 cells in the presence of this compound was found to be concentration-dependent. The isolated isoflavone glucoside at a low concentration resulted in lower cell proliferation activity than those in the presence of genistein and daidzein. Genistein at 0.3 ^M promoted cell proliferation by up to 1.99-fold over untreated cells (control), whereas the isoflavone glucoside at the same concentration caused the cells to proliferate by up to 1.14fold compared with control. However, at 5 ^.M, the isolated isoflavone glucoside increased cell proliferation by up to 1.74-fold over the control. This was nearly equal to that of genistein. Maximum cell proliferation was found to be in the presence of 5 ^.M daidzein. In addition, the isolated isoflavone glucoside, like daidzein and genistein, did not exhibit cytotoxicity to the MCF-7 cell line up to 50 ^.M (data not shown).

In this experiment, cell proliferation in the presence of daidzein was lower than that in the presence of genistein. This is in contrast to the results obtained by Han et al. (2002). It is known that the behavior and response of MCF-7 cells to 17ß-estradiol varies among different laboratories. The lack of a standard protocol, differences in conditions and MCF-7 strains in different laboratories may lead to significant inter-laboratory variability (Jones et al. 1998; Payne et al. 2000).

I _ ■


1—? 1 "f * f

T I" f J

- daidzin

- genistin HA

concentration (mm)

concentration (mm)

Figure 1. Estrogenic activity of phytoestrogens

- daidzin

- genistin HA

To confirm whether the estrogenic activity of the new isoflavone glucoside was due to its binding to the ER, the cells were incubated with various concentrations of the tested compounds in the presence of 1.5

^.M tamoxifen, an estrogen antagonist. Figure 2 shows the effect of the addition of tamoxifen on the estrogenic activity of the tested compounds. As shown in the result, cell proliferation in the presence of the tested compounds at all concentrations was blocked by tamoxifen.

Since cell proliferation in the presence of the isolated isoflavone glucoside was blocked by tamoxifen, it is suggested that the estrogenic activity was due to the binding of the isoflavone glucoside to the classical ERa. In addition, the structural similarity between the isolated isoflavone glucoside and the soy isoflavones, genistein and daidzein, suggests that they may have identical mechanisms of action.

0.01 0.1 concentration (^M)

0.01 0.1 concentration (^M)

Figure 2. Inhibition of estrogenic activity by tamoxifen (ER antagonist) Acetylcholinesterase activity

Acetylcholinesterase activity (AChE) is known as a marker of the neuronal differentiation in the rat pheochromocytoma cell line PC12. Results in Figure 3 show that 5 ^.M of isoflavone glucoside and daidzein significantly enhanced the AChE activity of PC12 cells in the presence of a low concentration of NGF (<0.1 ^M). At higher NGF concentrations (>20 ^.M), the AChE activity was slightly enhanced by 5 ^.M of the isoflavone glucoside and daidzein. However, the AChE activity in the presence of genistein was similar to that of the control under 0 to 50 ^.M NGF treatment conditions. Results in Figure 1 clearly indicate that the three test compounds are estrogenic, as shown in the E-screen assay results. The results further show that this estrogenic property is attributable to the binding of the phytoestrogen to the ER, since the effect was effectively blocked by tamoxifen (Figure 2).

In a previous study, Isoda et al. (2002) determined whether the enhanced AChE activity of PC12 cells by phytoestrogen was due to the binding of estrogenic compounds to the ER. They incubated the cells with varying concentrations of 17 P-estradiol (E2) and E2 plus genistein and daidzein or tamoxifen. The results showed that there is no significant enhancement of AChE activity under these conditions. This suggests that binding of E2 to the ER does not enhance AChE activity, and that possibly the effect of genistein and daidzein on AChE activity cannot be expressed in the presence of E2, considering that it requires 1,000- to 10,000-fold molar excess of these phytoestrogens to compete with the ability of E2 to bind to the ER (Miksicek RJ, 1993). From these results, the authors hypothesize that the enhanced AChE activity of PC12 cells in the presence of 5 ^M of the novel isoflavone glucoside may possibly be due to tyrosine protein kinase inhibition.

0.1 1 10 100 NGF concentration ing/ml )

Figure 3. Sensitivity of PC12 cells to NGF

To examine possible mechanisms of action, proteome analysis was performed. Figure 4 shows a typical CBB-stained 2D gel showing the protein profile of NGF-differentiated PC12 cells treated with daidzein. Spots of interest from comparable 2D gels are shown. Spots D1, D2, D3 and D4 are overexpressed in daidzein-treated PC12 cells, while spots C1 and C2 are underexpressed in daidzein-treated cells. The next step in this ongoing study is the excision of the spots and their identification through mass spectrometry. Novel proteins whose expressions are affected by phytoestrogens are expected to be characterized and identified at the end of the study.

Figure 4. Protein profile of NGF-differentiated PC12 cells incubated with daidzein

4. References

Deodato B, Altavilla D, Squadrito G, Campo GM, Arlotta M, Minutoli L, Saitta A, Cucinotta D, Calapai G, Caputi A P, Miano M & Squadrito F (1999) Cardioprotection by the phytoestrogen genistein in experimental myocardial ischaemia-reperfusion injury. Br J Pharm 128: 1683-1690.

Han DH, Denison MS, Tachibana H & Yamada K (2002) Relationship between estrogen receptor-binding and estrogenic activities of environmental estrogens and suppression by flavonoids. Biosci Biotechnol Biochem 66:1479-1487.

Isoda H, Talorete TPN, Kimura M, Maekawa T, Inamori Y, Nakajima N and Seki H (2002) Phytoestrogens genistein and daidzin enhance the acetylcholinesterase activity of the rat pheochromocytoma cell line PC12 by binding to the estrogen receptor. Cytotechnology 40: 117-123.

Jones PA, Baker VA, Irwin AJE & Earl LK (1998) Interpretation of the in vitro proliferation response of MCF-7 cells to potential estrogens and non-estrogen substances. Toxicol in Vitro 12: 373-382.

Knight DC & Eden JA (1996) A review of the clinical effect of phytoestrogens. Obstet Gynecology 87: 897-904.

Lamartiniere CA, Moore JB, Brown NM, Thompson R, Hardin MJ & Barnes S (1995) Genistein suppresses mammary cancer in rats. Carcinogenesis 16: 2833-2840.

Miksicek RJ (1993) Commonly occurring plant flavonoids have estrogenic activity. Mol Pharmacol 44: 37-43.

Payne J, Jones C, Lakhani S & Kortenkamp A (2000) Improving the reproducibility of the MCF-7 cell proliferation assay for the detection of xenoestrogens. Science Total Environt 248: 51-62.

Tham DM, Gardner CD & Haskell WL (1998) Potential health benefits of dietary phytoestrogens: A review of the clinical, epidemiological, and mechanistic evidence. J Clin Endoc Metab 83: 2223-2235.

Soto AM, Justicia H, Wray JW & Sonnenschein C (1991) p-Nonyl-phenol: An estrogenic xenobiotic released from "modified" polystyrene. Environ Health Perspect 92: 167173.

Soto AM, Sonnenchein C, Chung KL, Fernandez MF, Olea N & Serrano FO (1995) The E-SRCEEN assay as a tool to identify estrogen: An update on estrogenic environmental pollutants. Environ Health Perspect 103: 113-122.

White R, Jobing S, Hoare S A, Sumpter JP & Parker MG (1994) Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology 135: 175-182.


Junkyu Han, Mitsuaki Akutsu, Terence P. N. Talorete, Toshiyuki Tanaka and Hiroko Isoda

Graduate School of Life and Environmental Sciences, University of TsukubaTennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan

Abstract: On the basis of transepithelial electrical resistance measurements, we found that capsaicin (100^M) treated-human intestinal Caco-2 cells show a momentary decrease in tight-junction (TJ) permeability followed by a complete recovery. We used proteome analysis to search for proteins that are associated with the recovery of TJ permeability in capsaicin-treated Caco-2 cells. A protein of relative molecular mass of 14 kDa was found to be expressed higher in capsaicin-treated cells than in nontreated cells. Mass spectrometry and sequence analyse revealed that the protein expressed significantly by capsaicin treatment was the ribosomal protein P2 was and its cDNA sequence was identical to that found in the human genome database. An increase in amount of cellular filamentous actin (F-actin) was shown after 8 h of incubation with capsaicin. It is reported that ribosomal protein P2 can activate elongation factor 2, which stabilizes F-actin filaments, and that the deploymerization of F-actin was associated with the decrement in TJ permeability. Consequently, these results suggest that ribosomal protein P2 plays an important role in the recovery of the TJ permeability in capsaicin-treated human intestinal Caco-2 cells.

Key words: Caco-2 cells; capsaicin; ribosomal protein P2; TJ permeability


Human intestinal Caco-2 cells have been widely used as in vitro models to evaluate the transport of absorbing water, ions, and nutrients across the intestinal epithelial barrier (Satsu et al., 2003). Tight-junction (TJ) permeability increase in Caco-2 cells is regulated by various factors, such as food factors (capsaicin, capsianoside, etc.) and chemicals (ealkylphenolic compounds, EDTA, etc.). Such factors can alter

epithelial transport and barrier function of human intestinal epithelial cells by various mechanisms (cytoskeletal reorganization, redistribution of ZO-1 and occludin, exertion of Rho A, Rac 1, and Cdc 42, etc.) (Han et al., 2002; Nusrat et al., 1995; Bruewere et al., 2004).

In the previous studies (Han et al., 2002; Isoda et al., 2001), we showed that the TJ permeability increase (decrease in transepithelial electrical resistance (TER)) in capsaicin-treated (45 min) human intestinal Caco-2 cells is through binding of capsaicin to a capsaicin receptor-like protein. We also suggested that the increase in TJ permeability upon capsaicin treatment occurs due to a cytoskeletal reorganization of actin filaments, particularly due to the decrease in the amount of filamentous actin (F-actin) in Caco-2 cells. In addition, heat shock protein 47 (HSP47), which is activated during capsaicin treatment, plays an important role as a secondary messenger in the increase in TJ permeability by capsaicin treatment. A preliminary experiment indicated that Caco-2 cells treated with 100 ^M capsaicin longer than the time of previous studies, showed a decrease followed by a recovery of TER values. The recovery of TER values after capsaicin treatment, even without removal of capsaicin, was rapid. This suggests that some proteins, whose expressions are enhanced by capsaicin, facilitate the recovery of TER values after the momentary decrease.

Tools such as two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) enable the study of cellular proteome analysis. Using these techniques, changes in protein expression profiles in capsaicin-treated human intestinal Caco-2 cells, particularly proteins related to the recovery of TJ permeability, can be determined.

In this study, we performed 2-DE and MS to determine the possible mechanism behind the recovery of TER after a momentary decrease in capsaicin-treated Caco-2 cells. We found that the expression of ribosomal protein P2 is enhanced by capsaicin treatment, and discussed its role in regulating the recovery of TJ permeability.


TER measurement shows that the TJ permeability of the Caco-2 monolayer increased significantly (p < 0.05 vs control) upon treatment with capsaicin (100 ^M and 1 mM) (Figure 1). When treated with 100 ^.M capsaicin, the TJ permeability recovered after 60 min, followed by the complete recovery after 90 min. However, by treatment with 1 mM capsaicin, the TJ permeability increased irreversibly (Figure 1). These results suggest that the barrier function was disrupted by the high concentration of capsaicin (1 mM). However, treatment at low concentration (100 |M) resulted in the recovery of the barrier function. It indicates that the permeability of the intestinal TJ can be regulated by the low concentration of capsaicin (100 |M).

Time (min)

Figure 1. Effect of capsaicin on the TER. TER values areis presented as relative to those values at time zero time. The concentrations of capsaicin are: (open circles) 0 |M capsaicin; (filled boxes) 100 |M capsaicin; and (open triangles) 1 mM capsaicin.

To elucidate the mechanism behind the recovery of the barrier function, we used 2-DE and MS. The extracted proteins were first subjected to 2-DE (Figure 2 A). A number of proteins were found to be expressed more markedly in the capsaicin-treated cells than in the nontreated cells (data not shown). In particular, the protein spot indicated by arrows in Figure 2 B shows the highest expression in capsaicin-treated cells. The isoelectric point of this protein is in the range of 3.8-4.3, and it has a relative molecular mass of 14 kDa. To identify this protein, the spot was subjected to tryptic digestion and MALDI-TOF MS analysis. The protein spot has a sequence closest to that of ribosomal protein P2; the matched peptides cover 72% (83/115 of amino acids) of ribosomal protein P2.



45 min

90 mill


Figure 2. Two-dimensional 2D gel electrophoresis of nontreated-human intestinal Caco-2 cells (A), and time-dependent expression of a protein induced by capsaicin treatment (B). In panel (B), the encircled region in panel (A) and the corresponding region of the capsaicin-treated samples are magnified. The arrowed spot has an isoelectric point in the range of 3.8-4.3, and its molecular mass is approximately 14 kDa. 2-DE 2D gel electrophoresis was performed by the isoelectricon an focusing of proteins using immobilized pH 3-10 strips, followed by y the second-dimensional separation on 12-14% polyacrylamide gels. The separated proteins were stained with Coomassie brilliant blue (CBB). The encircled arrowed spot hais an isoelectric point in the range of present in the pH region 3.8-4.3 and its molecular mass is of 13-15 kDa.

By MS analysis, we were unable to get the complete amino acid sequence of the protein expressed by the capsaicin treatment. Therefore, we carried out the isolation and analysis of cDNA of ribosomal protein P2 from Caco-2 cells to examine whether there might be some discrepancy or diversity from the known nucleotide or amino acid sequence. The nucleotide and deduced amino acid sequences of the full-length cDNA clone of the ribosomal protein P2, from Caco-2 cell are shown in Figure 3. These sequences, and its 5'-upstream nucleotides sequence as well, are identical to those of the human ribosomal protein P2 deposited to the databases.

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Figure 3. Nucleotide and deduced amino acid sequences of ribosomal protein P2. An asterisk indicates a stop codon. The peptide fragments determined by MS analysis (Table 2) are indicated by lines under the sequences.

What is the relation between the recovery of the barrier function and the significant expression of ribosomal protein P2. Ribosomal protein P2 is a member of the well-conserved acidic P (phosphor) protein family in the eukaryotic ribosome (Gonzalo et al., 2002; Lavergne et al., 1987). It plays an important role in the elongation step of protein synthesis and in the activation of elongation factor 2 (EF-2) (Vard et al., 1997). It is reported that the EF-2 is activated by the homologous carboxy-terminal region of Ribosomal protein P2 (Furukawa et al., 1992). EF-2 interacts with globular actin (G-actin), stabilizes filament structure, and causes lateral association of F-actin (Bektas et al., 1994; Bektas et al., 1998). F-actin is a ubiquitous intracellular protein, which in filamentous form constitutes a major component of the cytoskeleton, and plays an essential role in cell motility and mechanics (Holmes et al., 1990; Dadabay et al., 1991). Moreover, it has been suggested that the cytoskeletal reorganization of actin filaments mediates the increase in TJ permeability (Lim et al., 2002). Therefore, it is possible that ribosomal protein P2, whose expression is enhanced by the capsaicin treatment, activates EF-2 to restore the F-actin in human intestinal Caco-2 cells followed by the prevention of the expressive TJ permeability. In the previous study (Isoda et al., 2001), we examined whether the capsaicin-induced increase in TJ permeability is associated with the cytoskeletal reorganization of the actin filaments by determining the cellular F-actin amount in Caco-2 cells by 45 min treatment of capsaicin (0, 200, and 300

^M). The amount of cellular F-actin was found to decrease significantly in the capsaicin-treated Caco-2 cells. This reduction is probably due to the depolymerization of F-actin into G-actin as a form of cytoskeletal reorganization (Rayment et al., 1993; Lim et al., 2002). To confirm the hypothesis, we determined the amount of cellular F-actin in Caco-2 cells treated with 100 ^M capsaicin with longer time scale (0, 8, 16, and 24 h), which is chosen to be comparable with the proteome analysis (Figure 2B). An increase in the amount of F-actin was shown in 8 h and continued till 24h of incubation (Figure 4). These and the previous results indicate that there is a close correlation between the amount of ribosomal protein P2 and that of F-actin.

Figure 4. Effect of 100 ^M capsaicin concentrations on the F-actin of Caco-2 cells.

Standard deviation were <10%.

In summary, we have investigated the mechanism of the recovery of TJ permeability in capsaicin-treated human intestinal Caco-2 cells. The expression of ribosomal protein P2 was enhanced by the capsaicin-treatment, which is followed by the increment in the amount of F-actin and the recovery of TJ permeability. This result suggests that ribosomal protein P2 stabilizes F-actin and recover the TJ permeability through the activation of EF-2. This is the first report showing that the expression of ribosomal protein P2 is related to the recovery of TJ permeability in human intestinal Caco-2 cells.


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