bk-2012-1109.ch018

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Chapter 18

Yerba Mate (Ilex Paraguariensis St. Hilaire) Saponins Inhibit Human Colon Cancer Cell Proliferation Sirima Puangpraphant,1 Mark A. Berhow,2 and Elvira de Mejia*,3 1Department

of Food Technology, Faculty of Science, Chulalongkorn University, Payathai Road, Patumwan, Bangkok, Thailand 10330 2USDA, ARS, National Center for Agricultural Utilization Research, 1815 N. University St., Peoria, IL 61601 3Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, 228 ERML, 1201 W. Gregory Drive, Urbana, IL 61801 *E-mail: [email protected]

Ilex paraguariensis St. Hilaire tree is native to South America and its dried leaves are used to prepare a traditional beverage called Yerba Mate tea. The aim of this study was to assess the anticancer properties of yerba mate saponins in vitro models. HT-29 (p53 mutant) and RKO (wild type p53) cells were treated with mate saponins (1- 200 μM). Mate saponins inhibited HT-29 (IC50 = 202 μM) and RKO (IC50 = 181 μM) cell proliferation. Mate saponins arrested HT-29 and RKO cells at G1 to S phase by significantly upregulating p21 and p27 proteins, and caused apoptosis through induction of Bax:Bcl-2 protein expression. Mate saponins induced apoptosis and cytotoxicity in human colorectal cancer cells independent of p53 status. Yerba mate tea saponins inhibit human colon cancer cell proliferation.

Introduction Saponins are a class of tritepenoid or steroid compounds that are widely distributed in plants, such as in soybean (1, 2), ginseng (3, 4) and yerba mate (5, 6). The primary saponins identified from Ilex paraguariensis were matesaponin 1 and 2 with ursolic acid as the triterpenoidal aglycone (Figure 1).

© 2012 American Chemical Society In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 1. (A) Aglycone of saponins (ursolic or oleanolic acids) with sugars attach at R, R1, R2, or R3 (B) LC-MS of an analytical run of yerba mate saponins.

Saponins from various plant sources affect the growth of colon cancer cells. Soy saponins prevent colon cancer by affecting cell morphology, cell proliferation enzymes, and cell growth of human colon cancer cells (7). Tin et al. (8) investigated the anti-carcinogenic effect of Astragalus saponins in HT-29 human colon cancer cells and in vivo using nude mice xenografts. They found that Astragalus saponins induced caspase-8 extrinsic apoptotic cascade and cause cell cycle arrest by modulation of both mTOR and ERK signaling pathways, of which inhibition of NF-κB was important in the latter mechanism (9). Puangpraphant et al. (10) demonstrated that saponins extracted from yerba mate dried leaves had anti-inflammatory activity in macrophages and inhibited HT-29 colon cancer cell proliferation by promoting apoptosis through activating caspase-3 activity. However, whether saponins from yerba mate affect the growth of cancer cells by causing apoptosis or necrosis or cell cyle arrest is still not clear. Colorectal cancer (CRC) involves a multistep pathway of colonic mucosal genetic mutations. Over 1.2 million new cases and 608,700 deaths were attributed 308 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


to CRC worldwide in 2008 making it the third and second most common cancer in males and females, respectively (11). One gene plays an important role in late CRC progression, the tumor suppressor gene, p53. Loss of p53 protein activity has been observed in 70-80% of colon adenocarcinomas suggesting that functional loss of p53 occurs late in tumorigenesis. Depending on the cell type and conditions, p53 regulates either cell cycle arrest or apoptosis. However, information about the mechanism of mate saponins on cell cycle arrest and mediators that regulate cell cycle, p21 and p27, and influence of p53 remains poorly understood. The aim of the present study was to determine the mechanism underlying the anti-colon cancer effects of yerba mate saponins in vitro. We evaluated the activity of yerba mate saponins on cell proliferation, apoptosis, and cell cycle regulations and focused on specific pathways mediated by p53 in human colon cancer cells.

Materials and Methods Chemicals and Cells Human colon cancer cell lines HT-29, RKO, normal colon fibroblast CCD-33Co, McCoy 5A medium, Eagle’s Minimum Essential Medium and 0.25% (w/v) Trypsin- 0.53 mM EDTA and Dulbecco’s Modified Eagle Medium with L-glutamine (DMEM) were purchased from American Type Culture Collection (Manassas, VA). Fetal bovine serum was purchased from Invitrogen (Grand Island, NY, U.S.A.). Bcl-2, Bax, p21, p27, p53 and actin mouse monoclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.) and antimouse IgG conjugated horseradish peroxidase secondary antibody was purchased from GE Healthcare (Buckinghamshire, U.K.). Cisplatin (> 99%) was purchase from Sigma (St. Louis, MO, U.S.A.). All other chemicals were also purchased from Sigma unless otherwise specified. Extraction and Purification of Yerba Mate Saponins Saponins were extracted and fractionated from yerba mate (Ilex paraguariensis) leaves, organically grown in Paraguay following the method as previously described (10). Confirmation of the identity of mate saponins in the mate extract fractions was performed by LC/ESI-MS analysis as previously described (10). Mate saponins were dissolved in DMSO at 10 mg/mL by weight as an initial stock and the initial stock was then diluted with culture medium to different concentrations (1- 200 μM) using the molecular weight of saponins equal to 1,000 to calculate μM equivalent concentrations. HT-29, RKO, and CCD-33co Cell Culture and Proliferation Assay HT-29 and RKO cells were cultured in McCoy 5A growth medium containing, 1% penicillin/streptomycin, 1% sodium pyruvate and 10% fetal bovine serum at 37 °C in 5% CO2/95% air. CCD-33Co colon fibroblasts were cultured in Eagle’s Minimum Essential Medium containing 10% FBS and 1% penicillin/streptomycin. A cell proliferation assay was performed using the MTS/PES CellTiter 96 Aqueous 309 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


assay kit (Promega Corporation, Madison, WI, U.S.A.) as previously indicated (12). For CCD-33Co, 1 × 103 cells per well were seeded in a 96-well plate and allowed to grow to confluence for one week with replacement of medium every other day. For HT-29 and RKO, 5x104 cells per well were seeded in a 96-well plate and total volume was adjusted to 200 μL with growth medium and allowed to grow for 24 h. Both cells were then treated with different concentrations of yerba mate saponins (1- 300 μM), caffeine (≥ 99%), caffeic acid (≥ 98%), quinic acid (≥ 95%), chlorogenic acid (≥ 95%), quercetin (≥ 98%), ursolic acid (≥ 90%) (1- 300 μM), and mate tea extract (1- 300 μg/mL) for 24 h. The extraction method of mate tea was previously described (12). Cell Cycle Distribution of HT-29 and RKO Colon Cancer Cells Analysis of cell cycle distribution was performed using flow cytometry. Briefly, HT-29 and RKO cells were seeded at a density of 2 × 105 cells per well in a 6-well plate and allowed to grow for 24 h at 37 °C in 5% CO2/95% air. The cells were then treated with different concentrations of mate saponins ranging from 1 to 200 μM for another 24 h at 37 °C in 5% CO2/95% air. After treatment, cells were fixed overnight with 70% ethanol at 4 °C and stained with propidium iodide solution (0.1% v/v). Cell cycle distribution analysis was performed using a LSR II flow cytometer (BD Biosciences (San Jose, CA, U.S.A.) at excitation wavelength of 488 nm. Fluorescence emission was measured using a 695/40 nm band pass filter. A total of 20,000 events were collected for each sample. The analysis was performed in triplicate. Analysis of Apoptosis of HT-29 and RKO Colon Cancer Cells The apoptotic status of the HT-29 and RKO colon cancer cells was evaluated by determining the presence of phosphatidylserine on the cell membrane using an Annexin V-FITC apoptosis detection kit (Sigma-Aldrich, MO, U.S.A.) by flow cytometry. Briefly, 2 x105 cells per well were seeded in a 6-well plate and allowed to grow for 48 h at 37 °C in 5% CO2/95% air. The cells were then treated with mate saponins (1-100 μM) for 12 h at 37 °C in 5% CO2/95% air. After treatment, cells were washed with PBS twice, trypsinized and suspended in binding buffer at a concentration of 1 x 106 per mL. Five hundred microliters of treated and untreated cells were transferred into a plastic test tube and stained with 5 μL Annexin V-FITC and 10 μL propidium iodide (PI) solution for 10 min. PI staining was performed concomitantly with Annexin V-FITC staining to determine whether any DNA/nuclei were present in the colon cancer cells. The cells were analyzed immediately by LSR II flow cytometer (BD Biosciences, San Jose, CA, U.S.A.). The analysis was performed in triplicate. Western Blot Analysis of p21, p27, Bax, Bcl-2, and p53 Protein Expression HT-29 and RKO cells were seeded at a density of 2 × 105 cells per well in a six-well plate for 24 h at 37 °C in 5% CO2/95% air. After 24 h incubation, cells were treated with mate saponins (1- 200 μM) for 24 h. After treatment, cells were 310 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

trypsinized and suspended in lysis buffer composed of 62.5 mM Tris–HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue, 5% β-mercaptoethanol and protease inhibitor cocktail (Thermo Scientific, Rockford, IL). Cell suspension was then used for Western blot for p21, p27, p53, actin, Bax and Bcl-2 using antibodies (1:200).


Statistical Analysis Data are presented as means ± SD for the indicated number of independently performed experiments. Data were analyzed using one-way ANOVA and means were considered to be significantly different at p < 0.05 as determined by Tukey’s.

Results Effect of Yerba Mate Saponins on Cell Proliferation of RKO Colon Cancer Cells Figure 2 shows that mate saponins inhibited RKO colon cancer cells proliferation in a concentration-dependent manner.

Figure 2. Dose-response curve of mate saponins on proliferation of RKO cells. Means with different letters are significantly different from each other (n = 3, p < 0.05).

The concentration of mate saponins that inhibit proliferation of HT-29 (IC50 = 201.8 μM) and RKO (IC50 = 181.0 μM) are shown in Table I. Mate saponins, mate tea extract and its constituents did not cause any cytotoxicity to CCD-33co normal colon fibroblast up to 300 μM (data not shown). The inhibition of proliferation in RKO (wild-type p53) was greater than with mate saponins-treated HT-29 (mutated p53). As shown in Table I, mate tea extract, chlorogenic acid, caffeic acid, quinic acid, and caffeine had a weak inhibition to both HT-29 and RKO cells. We also 311 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


measured cytotoxicity of cisplatin (> 99%) which is a chemotherapeutic drug to HT-29 (IC50 = 80.6 μM) and RKO (IC50 = 68.5 μM). We found that ursolic acid was significantly the strongest anti-proliferative agent to both colon cancer cells, HT-29 (IC50 = 30.2 μM) and RKO (IC50 = 68.5 μM).

Figure 3. Cell cycle distribution (%) of cells treated with yerba mate saponins in (A) HT-29 and (B) RKO. Means with different letters are significantly different from each other (n = 3, p < 0.05).

Yerba Mate Saponins Induced G1 Cell Cycle Arrest of HT-29 and RKO Cells The effects of mate saponins on cell cycle progression were studied by flow cytometry. For HT-29, p53-deficient cells, mate saponins (100 μM) significantly increased cells in G1 and decreased cells in S phase, resulting in an overall G1 to S-phase arrest (Figure 3A). Mate saponins (200 μM) also caused an increase of G2/M phase. For RKO, p53-proficient cell line, mate saponins affected cell cycle by significantly arresting at G1 to S-phase (Figure 3B). 312 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Effect of Yerba Mate Saponins on p21 and p27 Protein Expressions in HT-29 and RKO Cells


In order to investigate the mechanism by which mate saponins inhibit the growth of HT-29 and RKO colon cancer cells, we analyzed the protein expression of p21 and p27. Mate saponins significantly increased p21 protein expression in HT-29 at 200 μM (Figure 4A) and RKO at 100 μM (Figure 4B) and upregulated p27 protein expression in HT-29 at 1 μM (Figure 4C) and RKO at 200 μM (Figure 4D), suggesting that yerba mate saponins inhibit the cell proliferation by inducing cell cycle arrest.

Table I. Yerba mate tea bioactive compounds on human colon cancer cells Compound

HT-29 (µM)

RKO (µM)

*IC30

*IC50

IC30

IC50

Ursolic acid

16.2

30.2c

13.5

29.5d

Cisplatin

44.0

80.6b

40.2

68.5c

Quercetin

53.2

81.5b

83.2

189.4a

Saponins

82.3

201.8a

57.5

181.0b

Mate tea extract (µg/mL)

204.2

>300

198.2

>300

Caffeine

200.2

>300

200

>300

Chlorogenic acid

>300

>300

>300

>300

Caffeic acid

>300

>300

180

>300

Quinic acid

>300

>300

150

>300

* IC30 and IC50 are the concentrations (μM) that resulted in 30%, and 50% inhibition of cell proliferation (mean ± SD, n = 2). Different letters indicate significant differences, comparing differences within column, p < 0.05.

Yerba Mate Saponins Increased Apoptotic HT-29 and RKO Cells by Upregulating Bax:Bcl-2 Protein Expression To determine whether the cell death of HT-29 and RKO was due to apoptosis, we treated RKO cells with mate saponins at 50 and 100 µM for 24 h. Figure 5 shows that mate saponins treatment led to significant increase on apoptotic HT-29 and RKO cells.

313 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 4. Effect of different concentrations of mate saponins on protein expression of p21 in (A) HT-29 (B) RKO, and of p27 in (C) HT-29 and (D) RKO were assessed by Western blots. Actin was used as a protein loading control. The data represent the mean ± SD of a triplicate from three independent experiments. Different letters indicate significant differences, p < 0.05.



Figure 5. Effect of mate saponins on apoptotic (A) HT-29 cells; (B) RKO cells.



Mate saponins at 100 μM increased apoptotic cells from 4.7% (control) to 14.7% in HT-29, and from 2.0% (control) to 9.1% in RKO. We then analyzed induction of apoptosis by assaying the protein expression of apoptosis mediators, Bax and Bcl-2. Figure 6 shows that mate saponins significantly increased the ratio of Bax/Bcl-2 expression in RKO cells at 1 µM.

Figure 6. Effect of different concentrations of mate saponins on (A) protein expression of ratio Bax/Bcl-2 in RKO assessed by Western blots. Actin was used as a protein loading control. The data represent the mean ± SD of a triplicate from three independent experiments. Different letters indicate significant differences, p < 0.05.

Effect of Yerba Mate Saponins on p53 Protein Expression in HT-29 and RKO Cells Figure 7 shows that mate saponins at 1 μM significantly induced p53 expression in HT-29 (Figure 7A) but no significant changes were observed in RKO cells (Figure 7B).

Discussion Finding a compound that would work both in p53 mutated and wild-type cancer cells would be very useful, because approximately 50% of cancer cells are p53 mutated and the other half are p53 wild type. Also, inactivation of p53 causes 316 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


resistance to various cancer therapies including the use of an angiogenesis inhibitor (13) and 5-fluorouracil (14). The effect of mate saponin on p53 wild-type colonic cancer cells has not been reported. Thus, in the current study, we investigated the effects of mate saponins on cell growth and cell cycle arrest in p53 wild type colonic adenocarcinoma RKO cell lines which is known to be DNA mismatch repair defective, and p53 mutated colonic adenocarcinoma HT-29 cell lines. We found that ursolic acid is the strongest anti-proliferative bioactive compound to both colon cancer cells. Ursolic and oleanolic acids have been shown to have a protective effect against colon carcinogenesis in vivo (15). Mate saponins are amphiphilic compounds and categorized as triterpenoic saponins as soy saponins which have been shown to be able to interact with the cancer cell membranes that are rich in phospholipids and cholesterol and with the hydroxyl groups on the aglycone moiety (16). Our results showed that chlorogenic acid has a weak inhibition to both HT-29 and RKO cells which is consistent with Park et al. (17) who indicated that chlorogenic acid did not protect against AOM-induced tumorigenesis. Moreover, we found that cisplatin inhibited HT-29 and RKO cell proliferation with IC50 values consistent with published data (18–21). Yerba mate saponins reduced cell growth and caused cell cycle arrest in both cell lines. The influence of mate saponins on p21 was studied because of its suggested critical role in suppressing cell growth. Cell cycle is regulated by the activity of cyclin/cyclin-dependent kinase (CDK). This cyclin–CDK complex is regulated by CDK inhibitors such as p21 and p27. Mate saponins caused a dose-dependent increase in the expression of p21 and p27 in both HT-29 and RKO cells; p21 is well known as a p53 response gene capable of inhibiting multiple CDKs, resulting in the induction of G1 or G2 cell cycle arrest. Our results clearly demonstrated that G1 arrest via p21, by mate saponins, was through the upregulation of p53. Mate saponins increased the amount of RKO and HT-29 cells undergoing apoptosis in a concentration-dependent manner. Members of the Bcl-2 family of proteins are critical regulators of the apoptotic pathway (22, 23). These proteins consist of the major anti-apoptotic proteins, Bcl-x (L) and Bcl-2, and the major pro-apoptotic proteins Bax and Bak. Bax controls mitochondrial permeability and cytochrome c expression, and the release of cytochrome c from mitochondria to the cytoplasm is a key step in the initiation of apoptosis. As a downstream product of cytochrome c, caspases are critical mediators of the principal factors found in apoptotic cells (24). In the present study, mate saponins inhibited colon cancer cells proliferation by inducing apoptosis through increasing Bax/Bcl-2 ratio. These findings suggest that apoptosis induction in mate saponins-treated RKO cells involves the activation of the mitochondrial pathway. Consistent with our previous study, HT-29 cells treated with mate saponins resulted in a dose-dependent decrease in the anti-apoptotic Bcl-2 protein and increase in the expression of pro-apoptotic Bax protein (10). Our findings suggest the possible value of mate saponins against human colon cancer by promoting apoptosis of cancer cells.



Figure 7. Effect of different concentrations of mate saponins on protein p53 expression in (A) HT-29 and (B) RKO assessed by Western blots. Actin was used as a protein loading control. The data represent the mean ± SD of a triplicate from three independent experiments. Different letters indicate significant differences, p < 0.05.



Figure 8. Proposed mechanism through which mate saponins induce apoptosis and cell cycle arrest via upregulating p53.

Cellular stress and DNA damage typically trigger the p53 tumor suppressor gene to mediate a series of antiproliferative strategies by inducing both cell cycle arrest and apoptosis. One important link between p53 and apoptosis is based on the transcriptional control of proapoptotic members of the Bcl-2 family, such as Bax. The relationship between p53 protein and the HT-29 cell death is still not clear (25). Shen et al. (26) have found that 2’-OH flavanone inhibits the growth of HT-29 cells via increasing the expression of p21, but it has no effect on p53 protein. Tsai et al. (7) did not find any inhibitory effect of soy saponin on the p53 protein of WiDr cells (wild type p53 human colon cancer cells), consistent with our results. The p53 protein expression was not affected by mate saponins treatment in RKO cells, which contain wild-type p53 protein but mate saponins induced p21 in this cell line. These results indicate that wild-type p53 is not involved in the mate saponins-induced apoptosis in colon cancer cells. This mechanism of mate 319 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


saponins action is independent of the status of the p53 tumor suppressor gene. In addition, mate saponins still induced apoptosis in HT-29 cells, which contain mutant p53. We found that mate saponins could enhance the p53 cascade and prevent the expansion of mutated epithelial cells. Understanding the mechanism by which mate saponin induces cell cycle arrest in colonic adenocarcinoma cells has the potential of providing information needed to prevent or reduce the growth of colonic tumors. This might provide an understanding of how mate saponins reduce colonic epithelial cell growth in vitro. Further study of yerba mate saponins on tumor growth in vivo is yet still needed. An explanation for this mechanism of mate saponins in vitro and in vivo may guide rational approaches for preventing colonic carcinogenesis in humans. In summary, we proposed the mechanism by which yerba mate saponins inhibit HT-29 and RKO colon cancer cell proliferation by induction of cell cycle arrest and apoptosis via p53 cascade (Figure 8). Yerba Mate saponins arrested G1 cell cycle by inducing p21 and p27 CDK inhibitors. Mate saponins induced mitochondrial apoptosis by increasing the expression of the pro-apoptotic protein Bax, and decreased the expression of anti-apoptotic protein Bcl-2, resulting in an increase in caspase-3 activity. In addition, the ability of mate saponins to suppress cell growth of colonic tumorigenic cells was independent of the p53 status of the cells. This eliminates the need to screen tumorigenic colonic tissue for p53 status before treatment with mate saponins. Our findings suggest the possible value of mate saponin against human colon cancer by inducing cell cycle arrest and promoting apoptosis; mate saponins might be an effective agent in the prevention of CRC.

Acknowledgments Las Marias Company provided partial funds for this research and The Royal Thai Government a Scholarship to author SP. The authors declare no conflicts of interest.

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