I.INTRODUCTION

People usually drink coffee to enjoy the leisure time or to get a break from busy schedules or randomly at any time of the day. Either way, we should drink with caution and in moderate amounts, from a medical point of view. Recent results show that coffee consumption was associated with the lowering risk of several cancers, including liver, colorectal, mouth and throat, and breast cancer^2-5). However, there appeared wide controversies about the biological effect of coffee elements, and many authors insisted that coffee drinking would differently affect individual health depending on its dose^6-8).

In the previous study, the dialyzed coffee extract (DCE), up to 40 μg/mL, showed no harmful effect on RAW 264.7 cells for 12 hours, rather it produced stimulatory effect on RAW 264.7 cells by increasing the cell number and enhancing the protein expression of β-actin and Ki-67¹⁾. Therefore, it was confirmed that the DCE containing most low molecular elements of coffee extract was applicable to cell culture experiment without complicated purification procedures. Thereby, it was suggested that RAW 264.7 cells can be cultured with different dose of DCE, which are the equivalent to 2.5 cups (10 μg/mL), 5 cups (20 μg/mL) and 10 cups (40 μg/mL) of coffee for this study.

Further biological investigation was performed in this study using the RAW 264.7 cell culture system to know the molecular mechanism of the cellular proliferation induced by DCE. And the protein expression changes of RAW 264.7 cells were assessed depending on the dose of DCE through precision protein expression method, immunoprecipitation high performance chromatography (IP-HPLC). As the accuracy of IP-HPLC has been approved to produce the data of protein expression level in less than 5% error range, the molecular signaling mechanism of DCE effect in RAW 264.7 cells could be assessed in this study.

This study was aimed to elucidate the expression changes of proliferation-related proteins by different dose of DCE in RAW 264.7 cell culture. This study were statistically analyzed and discussed with reference to the published literature.

II.MATERIALS and METHODS

Dialyzed coffee extract (DCE) production and treatment

The coffee beans (Coffea arabica L.) were roasted and treated with hot water to get the ordinary coffee drink, followed by the dialysis using cellulose bag (131492, Spectra/ Por, Spectrum, CA, USA) which filtrates small molecules less than 1000 Da as done in the previous study¹⁾. The dose of dialyzed coffee extract (DCE) equivalent to 2.5 cup of coffee (DCE-2.5), DCE-5, and DCE-10 was separately added in the culture medium of RAW 264.7 cell culture.

RAW cell culture treatment with dialyzed coffee extract (DCE)

RAW 264.7 cells (ATCC, USA), murine macrophage cell line, were cultured in Dulbecco’s modified Eagle’s medium (WelGene Inc. Korea) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (WelGene Inc. Korea), 100 unit/mL penicillin, 100μg/mL streptomycine, 250ng/mL amphotericin B (WelGene Inc. Korea), at 5% CO2, 37.5°C. The cells were tested for mycoplasma on a regular basis to ensure that only mycoplasma-free cell lines were studied in the assays.

During the active growth of RAW 264.7 cells, the experimental groups were treated with DCE-2.5, DCE-5, and DCE-10 for 12 hours separately, while the control group was treated with normal saline. After the cell culture, the RAW 264.7 cells were observed under phase contracts inverted microscope, and their digital images (x400) were captured by a digital camera (DP-70^®, Olympus Co., Japan) and followed by statistical analysis using image analyzing program of IMT i-solution (ver 21.1, Vancouver, Canada).

Immunoprecipitation HPLC analysis for the protein extract obtained from RAW 264.7 cell culture

After 12 hours culture of RAW 264.7 cells treated with dialyzed coffee extract (DCE) equivalent to 2.5, 5, and 10 cups of coffee, DCE-2.5, DCE-5, and DCE-10, respectively, the RAW 264.7 cells were harvested with protein lysis buffer (0.3% SDS, 50 mM Tris-HCl pH 8.0, 0.3% β-mercaptoethanol, 1 mM PMSF, 1 mM EDTA) containing protein inhibitor cocktail (Sigma, USA). Then, the protein extracts were preserved in -70°C deep freezer to prevent further protein degradation.⁹⁾

100μg of protein extract was applied to each immunoprecipitation procedure using protein A/G agarose column (Amicogen, Korea). The protein A/G agarose columns were separately pre-incubated with 1μg of each 25 different antisera, including β-actin, Ki-67, PCNA, MAX, cMyc, E2F-1, Rb-1, and MAD (SantaCruz Biotech. USA). Briefly, the protein samples were mixed with 5 mL binding buffer (150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF and 0.5% NP-40), and incubated in the protein A/G agarose columns at 10°C for 1 hour. The columns were placed on the rotating stirrer during the incubation time. After washing each column with sufficient amount of PBS solution (pH 7.3, 137 mM NaCl, 2.7 mM KCl, 43 mM Na2HPO4-7H2O and 1.4 mM KH2PO4), the target protein was eluted with 150 μL IgG elution buffer (Pierce, USA). The immunoprecipitated proteins were analyzed by HPLC (1100 series, Agilent, USA) having a reverse phase column and micro-analytical detector system (SG Highteco, Korea), running with 0.15M NaCl, 20% acetonitryl solution at 0.4 mL/min for 30 min, and analyzed by UV spectroscope at 280 nm. Both control and experimental groups were simultaneously performed for the IP-HPLC. In the results of IP-HPLC, the sample protein peak areas (mAU*s) obtained from HPLC analysis were eliminated the antibody peak area (mAU*s) in the negative control, and recalculated into square root value to compare their protein expression levels between the experimental and control groups. All the square root values of protein peak areas were plotted into a graph (Fig. 3 and 4) depending on the characteristic protein groups.¹⁰⁾

III.RESULTS

RAW 264.7 cells, immortalized murine macrophages, treated with DCE showed different expressions of cellular proliferation-related proteins depending on the dose of DCE, i.e., DCE-2.5, DCE-5, and DCE-10. In the histological observation of RAW 264.7 cells treated with DCE for 12 hours, the average cell number shown in high magnification (×400) increased up to 157.7±15.34 by DCE-2.5 and 150.2±9.03 by DCE-5, but decreased by DCE-10 (116.3±12.76) to the level of the control (112.5±13.3) (Fig. 1). Many RAW 264.7 cells treated by DCE-2.5 and DCE-5 became round to polygonal in shape, implying the frequent mitotic figures, while RAW 264.7 cells treated with DCE-10 showed dendritic cytoplasm processes similar to the cells in the control group.

In the IP-HPCL analysis, the proliferation-related proteins, such as PCNA, Ki-67, and MPM2 showed consistent up-regulation by DCE 2.5 and DCE-5, but there was sparse proliferation effect by DCE-10 compared to the control. The cellular proliferation effect of DCE was the highest in the DCE-2.5 treatment, and gradually decreased in DCE-5, but it became similar to the control level in DCE-10. On the other hand, a proliferation inhibitor, p21 was decreased in DCE-2.5 but gradually recovered to the control level in DCE-5 and DCE-10 treatment (Fig. 2).

Regarding the cMyc/MAX/MAD signaling, the RAW 264.7 cells treated with DCE showed the activation of proliferation (cMyc-MAX signaling) with the inactivation of anti-proliferation factor, MAD, in DCE 2.5 and DCE-5 treatment. These cMyc/MAX/MAD signaling were almost not affected by DCE-10. The co-expression of cMyc and MAX was more intense in DCE-5 treatment than in DCE-2.5 treatment, and the cMyc expression was reduced by the increased expression of MAD in DCE-10 treatment. These dynamic changes of cMyc/MAX/MAD protein expression indicated that the cMyc-related cellular proliferation dominantly occurred in DCE-5 treatment, which was subsequently suppressed in DCE-10 treatment (Fig. 3).

The p53/Rb-1/E2F-1 signaling was variable in the RAW 264.7 cells treated with different dose of DCE. RAW 264.7 cells treated with DCE showed the activation of E2F-1 by decrease of tumor suppressor protein, Rb-1 in DCE-2.5, while gradual inactivation of E2F-1 by increase of Rb-1 in DEC-5 and DCE-10. The expression of p53 was slightly increased in DCE-5 treatment, but subsequently recovered to the control level in DCE-10 treatment. These dynamic changes of p53/Rb-1/E2F-1 protein expression indicated that the E2F-1-related cellular proliferation dominantly occurred in DCE-2.5 treatment, which was gradually suppressed in DCE-5 and DCE-10 treatment (Fig. 4).

Taken together, the DCE-2.5 and DCE-5 induced the proliferation of RAW 264.7 cells in vitro, while DCE-10 produced sparse effect on the proliferation of RAW 264.7 cells. The essential proliferation-related proteins, E2F-1 and cMyc were differently expressed in RAW 264.7 cells depending on the dose of DCE. The RAW 264.7 cells showed the dominant expression of E2F-1 in DCE-2.5 treatment and the dominant expression of cMyc in DCE-5 treatment compared to the control, while they showed no dominant expression of both proteins in DCE-10 treatment.

IV.DISCUSSION

Although coffee drinking was known to give some beneficial effects on human health, the accumulated amount of coffee in the body is hardly measurable to control its antioxidative role in the individual people¹¹⁾. And more, the complicated coffee elements produced while coffee bean roasting are also not elucidated clearly so far. Therefore, in order to know the cellular effect of coffee extract in vivo, we made the DCE through simple dialysis method using cellulose bag to get the small elements less than 1000 Da, and the DCE was directly applied into the RAW 264.7 cell culture. The DCE may contain some coffee elements which are not freely absorbable through gastrointestinal tract in vivo, so that it is presumed that the DCE may induce some harmful effect on the cells in in vitro experiment. Nevertheless, the major part of DCE is expected to play the similar role in the cell culture in vitro to the ordinary coffee drink in vivo, which is expected to be absorbed freely from animal gastrointestinal tract in vivo. Therefore, it is assumed that the dialyzed coffee molecules may be appropriate for the application into in vitro cell culture. Drinking coffee has been known to enhance the immunity as well as cardiovascular stimulation and so on^12-14). For the immunological effects of dialyzed coffee molecules, the present study utilized the murine macrophage cell line, RAW 264.7, to perform the histological observation and different protein expression assays.

First of all, in the histological observation the DCE induce the proliferation of RAW 264.7 cells, resulted in the most increased cell number in DCE-2.5 treatment and followed by in DCE-5 treatment but almost similar cell number in DCE-10 treatment in the present study. These findings were also similar to the preliminary study done previously¹⁾.

In the IP-HPCL analysis the proliferation-related proteins, the cellular proliferation effect of DCE was the highest in the DCE-2.5 treatment, and gradually decreased in DCE-5, but it became similar to the control level in DCE-10. Particularly, the co-expression of cMyc and MAX was more intense in DCE-5 treatment than in DCE-2.5 treatment, and cMyc expression was reduced by the increased expression of MAD in DCE-10 treatment. These dynamic changes of cMyc/MAX/MAD protein expression may be implicative for the dominant cMyc-related cellular proliferation in DCE-5 treatment. As the cMyc/MAX/MAD network comprises a group of transcription factors whose distinct interations result in gene-specific transcriptional activation or repression ¹⁵⁾. In this study, the co-expressions of cMyc/MAX and cMyc/MAD were clearly distinguishable in the IP-HPLC anaylsis, thereby it could be defined that the cellular proliferation of RAW 264.7 was affected by the dynamic expression of cMyc signaling.

The p53/Rb-1/E2F-1 signaling was also variable in the RAW 264.7 cells depending on the dose of DCE. The activation of E2F-1 was associated with the decrease of tumor suppressor protein, Rb-1 in DCE-2.5, and the inactivation of E2F-1 was related to the increase of Rb-1 in DEC-5 and DCE-10. However, the expression of p53 was slightly increased in DCE-5 treatment, but subsequently recovered to the control level in DCE-10 treatment. These dynamic changes of p53/Rb-1/E2F-1 protein expression may be deeply implicative for the elevated E2F-1-related cellular proliferation in DCE-2.5 treatment.

The main finding of the present study cements the conclusion as shown in Fig.5. A scheme representing the dose dependent cellular effect of DEC in RAW 264.7 cells in vitro indicated that DCE-2.5 showed the most intense proliferative effect, and followed by DCE-5. These enhanced proliferations of RAW 264.7 cells, macrophage, might be related to the increase of innate immunity (Fig. 5). However, the additional investigation should be followed to explore more biological effect of DCE with precise molecular biological methods.

As mentioned previously, coffee extract was not usually considered to play specific interaction with certain signaling proteins, but the DCE induced remarkable changes in the expression of proliferatio-related proteins in this study. Especially, the DCE dose dependent change of cMyc and E2F-1 signaling was conspicuous in RAW 264.7 cells. This DCE dose dependent modality in protein expression may be caused by the biochemical properties of DCE elements, including caffein, chlorogenic acid, etc., which are small organic compounds interacting with various cellualr substances.

In summary, this study was performed to know the cellular effect of coffee drink using the dialyzed coffee extract (DCE). The RAW 264.7 cells treated with the amount equivalent to 2.5, 5, and 10 cups of coffee were cultured for 12 hours, and their protein extracts were analyzed by IP-HPLC. RAW 264.7 cells differently expressed their proliferation-related proteins depending on the dose of DCE-2.5, DCE-5, and DCE-10, and resulted that the DCE-2.5 and DCE-5 induced the proliferation of RAW 264.7 cells by increased expression of E2F-1 and cMyc, respectively, while DCE-10 produced sparse effect on the proliferation of RAW 264.7 cells.