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Jan 23, 2015 - Cell Death in Mouse Colon Cancer CT-26 Cells and Transplanted ... However, α-tomatine induced cell death in CT-26 cancer cells through...
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The Tomato Glycoalkaloid α‑Tomatine Induces Caspase-Independent Cell Death in Mouse Colon Cancer CT-26 Cells and Transplanted Tumors in Mice Sung Phil Kim,† Seok Hyun Nam,*,† and Mendel Friedman*,§ †

Department of Biological Science, Ajou University, Suwon 443-749, Republic of Korea Western Regional Research Center, Agricultural Research Service, U.S Department of Agriculture, Albany, California 94710, United States

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ABSTRACT: Tomatoes (Solanum lycopersicum) produce the bioactive glycoalkaloid α-tomatine. This study determined the effect of commercial α-tomatine on CT-26 colon cancer cells in vitro and in vivo in an intracutaneously transplanted mouse tumor. Cytotoxicity experiments showed that α-tomatine induces about 50% lysis of the colon cancer cells at 3.5 μM after 24 h of treatment. Large proportions of cells were found to be in the annexin V (+)/propidium iodide (+) phase of cell death, implying late phase apoptotic/necrotic status. However, α-tomatine induced cell death in CT-26 cancer cells through caspase-independent signaling pathways. This conclusion was supported by Western blot analysis showing a localization of apoptosis-inducing mitochondrial protein (AIF) to the nucleus and down-regulation of survivin (an inhibitor of apoptosis) expression as well as failure to detect the active form of caspase-3, -8, and -9 produced by proteolytic cleavage in CT-26 cancer cells. Intraperitoneally administered α-tomatine (5 mg/kg body weight) also markedly inhibited growth of the tumor using CT-26 cancer cells without causing body and organ weight changes. The reduced tumor growth in the mice by 38% after 2 weeks was the result of increased caspase-independent apoptosis associated with increased nuclear translocation of AIF and decreased survivin expression in tumor tissues. α-Tomatine in pure form and in tomatine-rich green tomatoes might prevent colon cancer. KEYWORDS: α-tomatine, tomatoes, CT-26 colon cancer cells, mouse tumor inhibition, body and organ weights, mechanism, research needs

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INTRODUCTION We previously reported that black and brown rice brans and the black rice bran compound γ-oryzanol inhibited implanted CT26 mouse cancer cell tumors1,2 and that Hericium erinaceous edible mushroom extracts inhibited the migration (metastasis) of the implanted CT-26 murine colon carcinoma cells to the lung,3 suggesting that these rice brans and mushrooms have the potential to serve as a health-promoting functional foods. Because the tomato glycoalkaloid α-tomatine is also reported to inhibit the multiplication of different cancer lines in vitro and in vivo, as described in some detail below, it was of interest to find out if this natural compound that is found in high amounts in immature (green) tomatoes,4−6 in wild tomato fruit,7 in a widely consumed high-tomatine red tomato variety grown in the Andes mountains of Peru,8 and in uncultivated potato accessions9 but in low amounts in red tomatoes10 would also inhibit growth of the transplanted colon tumors. Surprisingly, the α-tomatine content from organically and conventionally grown red tomatoes harvested over a 10 year period ranged from 4.3 to 111.8 μg/g dry weight, with the average value for organic tomatoes twice that of the conventional ones.11 The cited studies show that the tomatine content of fresh tomatoes ranged from about 4 to 42 mg/kg on a freeze-dried dry weight basis and that the levels of commercially available pickled green and fried green tomatoes are 50−100 times higher than those of the standard red varieties. Because one objective of the present study was to define the role of caspases in the inhibition of tumor growth, we will © 2015 American Chemical Society

briefly mention the reported roles of caspases in the prevention of carcinogenesis. Caspases are proteolytic enzymes that seem to mediate apoptosis via two types of feedback loops.12 The first involves activation via cleavage of the anti-apoptotic Bcl-2 and Bcl-xL proteins independent of caspase-3, and the second involves activation of caspase-3. In another positive feedback loop, caspase-3 can cleave the X-chromosome-linked inhibitor of apoptosis (XIAP) to further sensitize cancer cells to induced apoptosis. Ho et al.13 describe similar caspase-dependent pathways for caspase-3, -8, and -9. The objective of the present study is to compare the αtomatine-induced inhibition of CT-26 colon cancer cells in vitro and the growth of implanted tumors in a mouse model and to define the mechanism of the observed beneficial effects.

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MATERIALS AND METHODS

Test Compounds. DMEM, phosphate-buffered saline (PBS), fetal bovine serum (FBS), and other miscellaneous cell culture reagents were purchased from Hyclone Laboratories (Logan, UT, USA). Fluorescein isocyanate (FITC)-conjugated annexin V, propidium iodide (PI), and α-tomatine were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All reagents of analytical grade were purchased from Sigma Chemicals (St. Louis, MO, USA) and used without further purification. Received: Revised: Accepted: Published: 1142

August 21, 2014 December 22, 2014 January 12, 2015 January 23, 2015 DOI: 10.1021/jf5040288 J. Agric. Food Chem. 2015, 63, 1142−1150

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Journal of Agricultural and Food Chemistry Chemicals. We previously reported that the tomato glycoalkaloid referred to as tomatine consists of a 9:1 mixture of α-tomatine and dehydrotomatine.14 This ratio differs for different parts of the tomato plant, ranging up to 3:1 for tomatine isolated from large stems.15 The purity of the α-tomatine used in the present study as provided by the supplier is >75%. α-Tomatine (C50H93NO21; mol wt 1034.2) has the following chemical name: O-[β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3]-β-D-glucoyranosyl-(1→4)-β-D-galactopyranoside]. Dehydrotomatine has the same structure as α-tomatine except for the presence of a double bond in the steroidal B ring of the aglycone part of the molecule. α-Tomatine has a tetrasaccharide (lycotetraose) attached to the aglycone tomatidine, whereas dehydrotomatine has the lycotetraose moiety attached to the aglycone tomatidenol (Figure 1).

product. The absorbance of resultant colored products was read in a microplate reader (model 550, Bio-Rad, Hercules, CA, USA) at 490 nm. Cell cytotoxicity was expressed by the following formula: cytotoxicity (%) = 100 × (absorbance of α ‐tomatine‐treated cells /absorbance of maximum LDH‐released control cells)

The concentration that results in ∼50% cell death for 24 h treatment was 3.5 μM. We used this concentration and incubation time period to avoid any possible adverse effect of tomatine on the cells. In parallel, α-tomatine-induced cytotoxicity was also assessed by an MTT staining method as described in our previous publication.3 Apoptosis Assay by Flow Cytometry. Flow cytometry was carried out according to the method described by Vermes et al.17 Briefly, CT-26 cancer cells (1 × 106) were seeded into a culture dish and cultivated for 3 h in serum-free DMEM. Serum starvation is needed for all cells to synchronize their cell cycle. This step reduces the background noise of the data. After serum starvation, the serumfree medium was replaced with complete medium. Cells were then treated with 3.5 μM α-tomatine. After incubation for each time period, cells were collected by detaching through trypsin digestion and subsequent centrifugation at 1000g for 5 min. Cell pellets were resuspended in a FACS binding buffer (0.1 mL; 10 mM HEPES, 150 mM NaCl, 2.5 mM CaCl2, pH 7.4) with annexin V-FITC (1 μg) and PI (1 μg). After incubation for 15 min in the dark, flow cytometry was carried out in a FACSCaliber (Becton, Dickinson and Co., San Jose, CA, USA). Mice and Treatments. All experiments were performed in compliance with the relevant laws and institutional guidelines. Pathogen-free female BALB/c mice (6 weeks old), weighing 20−25 g, were purchased from Orient Bio (Seongnam, Korea). The mice were hosted in a stainless steel cage under a 12 h light/dark cycle with a temperature range of 20−22 °C and a relative humidity of 50 ± 10%. The mice were fed pelletized commercial chow diet from Orient Bio (catalog no. 5L79) and sterile tap water ad libitum during the entire experimental period. After acclimation for 1 week, the mice were then randomly divided into two groups (n = 10), avoiding any intergroup difference in body weight. α-Tomatine was dissolved in 0.1% DMSO. After dilution with PBS to an appropriate dose, α-tomatine was given to mice via the intraperitoneal (ip) route at a dose of 5 mg/kg body wt (in 200 μL). Simultaneously, the mice were intracutaneously transplanted with 1 × 106 of CT-26 cancer cells in 200 μL of PBS into the lateral side of the back. The mice were then administered the α-tomatine dose once a day for 2 weeks. Control mice were administered the same volume of vehicle only. Mice were sacrificed by CO2 inhalation at the end of the experimental periods for excision of tumor masses. Preliminary experiments indicated that the mice frequently died 4 weeks after tumor transplantation, especially the vehicle-treated control mice without tomatine. To avoid this problem, we carried out the in vivo experiment for 2 weeks. Tumor Growth. To evaluate the suppressive effect of α-tomatine on tumor growth, removed tumor masses from the control and experimental groups of mice were weighed using an analytical balance. Excised tumor masses were rinsed with ice-cold PBS and stored at −20 °C until use. Caspase-3, -8, and -9 Activity Assay. Caspase activities were measured using the caspase-3, -8, and -9 activity kits purchased from Millipore (Billerica, MA, USA) according to the manufacturer’s instructions. Briefly, α-tomatine-treated cells (1 × 106 cells) or tumor tissues were extracted in an ice-cold lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% NP-40, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM PMSF, 10 μg/mL aprotinin, 10 μg/ mL leupeptin) for 30 min, and pulverization of tumor tissues using a homogenizer was conducted for effective protein extraction in a lysis buffer. Lysates were clarified by microcentrifugation at 14000g for 10 min. Supernatants (cytosolic extract) containing proteins (50 μg) were incubated in an assay buffer (100 μL) containing colorimetric substances, Ac-Asp-Glu-Val-Asp (DEVD)-p-nitroaniline (pNA), AcIle-Glu-Thr-Asp (IETD)-p-NA), and Ac-Leu-Glu-His-Asp (LEHD)-

Figure 1. Structures of α-tomatine and dehydrotomatine. In αtomatine, the tetrasaccharide (lycotetraose) side chain is attached to the aglycone tomatidine and in dehydrotomatine, to the aglycone tomatidenol. α-Tomatine is soluble in methanol, ethanol, dioxane, and dimethyl sulfoxide (DMSO) and is fairly soluble in water. The experiments were carried out with solutions of 0.1% commercial tomatine in DMSO. This concentration is known not to induce cell toxicity. The term “tomatine” describes a mixture of α-tomatine and dehydrotomatine. Cancer Cells and Mice. The CT-26 mouse colon cancer cell line from the American Type Tissue Culture Collection (Manassas, VA, USA) was cultured in DMEM supplemented with 10% heatinactivated FBS containing penicillin (100 U/mL) and streptomycin (100 mg/mL). Cells were cultured at 37 °C in a humidified atmosphere with 5% CO2. Cytotoxicity Assay. Cell cytotoxicity was assessed by lactate dehydrogenase (LDH) release upon cell lysis following the method of Nachlas et al.16 A CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega Corp., Madison, WI, USA) was used following the manufacturer’s instructions. Briefly, the CT-26 cancer cells were seeded into a 96-well plate at a density of 1 × 105 cells/well and cultured for 24 h in 37 °C humidified air with 5% CO2. The cells were then treated with α-tomatine under various concentrations (from 1 to 10 μM final concentration) for various time periods (from 3 to 48 h). After treatments, the supernatant from each well was transferred to the flat-bottom 96-well enzymatic assay plate. Released LDH in culture supernatants was measured with a 30 min coupled enzymatic assay, resulting in the conversion of a tetrazolium salt into a red formazan 1143

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Journal of Agricultural and Food Chemistry

Figure 2. Cytotoxic effect of α-tomatine on CT-26 mouse colon cancer cells. (A) The cells were plated at a density of 1 × 105 cells/well in a 96-well microplate and then treated with the indicated concentrations of α-tomatine for 24 or 48 h at 37 °C, followed by measurement of cell death by LDH activity. (B) The cytotoxicity of α-tomatine was measured for the indicated time periods at a concentration of 3.5 μM. Plotted values are the mean ± SD (n = 3).

Table 1. Changes in Caspase-Independent CT-26 Mouse Colon Cancer Cell Death According to α-Tomatine Incubation Timesa cell viability (%) sample vehicle z-VAD-fmkb tomatinec tomatine + z-VAD-fmk

3h 100.0 102.6 98.7 99.8

± ± ± ±

6h 3.4 6.5 5.3 4.5

100.0 105.7 92.5 93.1

± ± ± ±

12 h 4.7ab 5.1a 3.3b 5.2b

100.0 108.4 71.6 74.0

± ± ± ±

24 h 2.7b 5.8a 3.6c 4.0c

100.0 115.3 59.3 61.9

± ± ± ±

5.6b 7.7a 3.1c 4.2c

Values are expressed as the mean ± SD of triplicate experiments. Values not sharing the same letter in the column are significantly different at p < 0.05. b100 μM z-VAD-fmk used as a pan-caspase inhibitor indicates z-Val-Ala-Asp-fluoromethyl ketone. c3.5 μM α-tomatine in 200 μL of DMSO was administered once a day. 100 μM z-VAD-fmk used as a pan-caspase inhibitor indicates z-Val-Ala-Asp-fluoromethyl ketone. Vehicle indicates 0.1% DMSO. MTT assay was employed to assess cytotoxicity. Cytotoxicity (%) = 100 × (1 − absorbance of α-tomatine-treated cells/absorbance of vehicle-treated cells). a

pNA for caspase-3, -8, and -9 activity assays, respectively, at 37 °C for 2 h. Absorbance at 405 nm was quantified in a microplate reader. Western Blot Analysis of Cell Proteins. α-Tomatine-treated CT-26 cancer cells (106 cells/mL) were harvested and then lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris Cl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA, pH 7.4) to prepare whole cell proteins. For the preparation of nuclear protein fraction, the cells were suspended in hypotonic buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, pH7.9) and were allowed to swell on ice. After the addition of NP-40 (final 0.6%), the cells were subjected to vigorous vortexing and centrifugation at 12000g. The resultant nuclear pellet was further extracted with hypertonic buffer (20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, pH7.9). The extract was centrifuged at 12000g to recover the supernatant termed nuclear fraction. Protein concentrations were determined according to the Bradford method using a Bio-Rad Protein kit. Bovine serum albumin (BSA) was used as standard. The cell extract containing proteins (30 μg) was separated on 12% SDS−polyacrylamide gels and electrophoretically transferred onto nitrocellulose membrane (Millipore). The membrane was blocked in 5% skim milk at 4 °C overnight and probed with the primary antibodies as follows: anti-p65 nuclear factor kappa B (NFκB) monoclonal antibody (catalog no. 6956S, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-β-actin monoclonal antibody (catalog no. 04-1116, Millipore), rabbit anti-mouse apoptosis-inducing factor (AIF) polyclonal antibody (catalog no. 4642S, Cell Signaling Technology, Inc.), anti-survivin monoclonal antibody (catalog no. 2808S, Cell Signaling Technology), 21 monoclonal antibody (catalog no. MAB10072, Millipore), and anticaspase-3 (catalog no. 9664S), anti-caspase-8 (catalog no. 9429S), and anti-caspase-9 polyclonal antibodies (catalog no. 9509S, Cell Signaling Technology). After allowing the primary antibody reaction for at least

3 h, the secondary antibody reaction with horseradish peroxidase (HRP)-conjugated anti-IgG antibody was performed under the same conditions. Blots were developed using the ECL detection kit (Pierce, Rockford, IL, USA). The intensity of separated protein bands was quantified using a gel documentation system (model LAS-1000CH, Fuji Photo Film Co., Tokyo, Japan). At least three separate replicates were determined for each experiment. Statistical Analysis. Results are expressed as the mean ± SD of three independent experiments. Significant differences between means were determined by ANOVA test using the Statistical Analysis Software package SAS (Cary, NC, USA). p < 0.05 is regarded as significant.

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RESULTS Growth Inhibition of CT-26 Cells. Figure 2 shows the cytotoxicity of α-tomatine on CT-26 mouse colon cancer cells. Treatment with α-tomatine decreased cell viability in a concentration- and time-dependent manner. The maximum inhibitions with α-tomatine at 10 μM were about 86.4 and 92.63% at the end of the 24 and 48 h treatments, respectively. The calculated concentration that results in ∼50% cell death after 24 h was 3.5 μM. Death Pattern of CT-26 Cells. Next, experiments were carried out to answer the question of whether or not the growth inhibition by α-tomatine resulted from apoptotic cell death. The 3.5 μM α-tomatine treatment for 24 h that induced 48% cell death was used to further treat CT-26 cancer cells. At this concentration, we examined whether the pan-caspase inhibitor,18 z-VAD-fmk, blocks α-tomatine-induced cell death. The results of the MTT assay (Table 1) show that cell death 1144

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Journal of Agricultural and Food Chemistry was 71.6 and 59.3% at 12 and 24 h of incubation, respectively. z-VAD-fmk (100 μM) failed to reverse α-tomatine-induced cell death; cell viability was not significantly different at p < 0.05 between tomatine-treated and tomatine plus the caspase inhibitor-treated groups. The results of the apoptotic cell death process on αtomatine-treated CT-26 cancer cells were confirmed by flow cytometric analysis using double staining with annexin V-FITC and PI. This method detects apoptosis by monitoring translocation from the inner membrane to outer cell surface.19 Indeed, FITC-labeled annexin V can bind to PS to detect apoptotic cells. Figure 3 shows that ∼32% of CT-26 cancer cells

Figure 3. Contour diagram of annexin V-FITC/PI flow cytometry of CT-26 cells after being treated with α-tomatine for the indicated incubation periods. After being treated with 3.5 μM α-tomatine, cells were treated with 1 μg each of annexin V-FITC and PI, respectively, for 15 min. The lower left quadrants of each panel show the viable cells, which exclude PI and are negative for annexin V-FITC binding. The lower right quadrants contain the early apoptotic cells, positive for annexin V-FITC binding and negative for PI uptake, whereas the upper right quadrants contain the later apoptotic/necrotic cells, positive for annexin V-FITC binding and for PI uptake. The figure represents three independent experiments.

treated with α-tomatine for 12 h were stained by annexin VFITC, which represented the early stage of apoptotic cells, and 16% of the cells were stained by both annexin V and PI, which indicated late-phase apoptosis/necrosis. After treatment for 24 h, 22% of the cells were in the early phase of apoptosis and 49% in the late phase. CT-26 Cell Death Is Independent of Caspase Activation. Caspase activation is known to play an important role in both intrinsic and extrinsic apoptotic pathways.20 Among caspases, activation of caspase-3, -8, and -9 is thought to be an important mechanism in the apoptosis pathway (caspase3 effector caspase, caspase-8 and -9 initiator caspases). Figure 4A shows that the α-tomatine (3.5 μM) treatment failed to increase the initiator caspase activities of caspase-8 and -9 to statistically significant levels. In contrast, the effector caspase-3 activities were significantly induced by α-tomatine treatment during the 12 and 24 h incubations (about 3.2- and 5.0-fold increases during 12 and 24 h incubations, respectively). However, the extent of activity increase was much less than that observed in 50 μM etoposide-treated positive control (18.8- vs 3.2-fold increase during the 12 h incubation). Etoposide used in the assay is a compound that induces cell apoptosis through the caspase-dependent signal transduction

Figure 4. Changes in caspase-3, -8, and -9 activities by treatment with α-tomatine for the indicated incubation times in CT-26 cells. (A) The enzymatic activities of caspase-3, -8, and -9 in the cell lysates were determined by incubation with caspase-specific colorigenic substrates DEVD-pNA, IETD-pNA and LEHD-pNA, respectively. (B) Active forms of caspases produced from pro-forms by proteolytic cleavage. Total cell lysate from α-tomatine-treated cells was subjected to Western blot analysis using caspase-3, -8, and -9 specific antibodies. βActin was used as an internal control. Etoposide-treated controls (50 μM) were subjected to incubation for 12 h. Bars not sharing a common letter are significantly different between groups at p < 0.05. The figures are representative of three independent experiments.

pathway. It is generally used as a positive control to determine whether cell apoptosis occurs by triggering of caspases. Because etoposide exerts its apoptogenic effect via a caspase-dependent pathway, these findings on α-tomatine-triggered caspase-3-, -8, 1145

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Journal of Agricultural and Food Chemistry and -9 activation modes suggested that CT-26 cell death by αtomatine largely occurs through a caspase-independent pathway. Western blot analysis was employed to further examine whether active forms of caspases were produced by proteolytic cleavage from pro-forms in α-tomatine-treated cells due to caspase-dependent apoptosis. Because no detectable protein bands of active forms of caspases were present in Figure 4B, this method confirms that α-tomatine-induced cell death most probably occurred through caspase-independent pathways. Inhibition of Survivin and NF-κB/p65 Expressions and Induction of AIF Nuclear Translocation. α-Tomatineinduced cell death independent of caspase activation was also supported by the following data (not shown). First, no 4′,6′diamidino-2-phenylindole (DAPI)-stained chromosomal condensation and apoptotic body appeared during the test conditions. Second, digestion of genomic DNA into a ladder of internucleosomal fragmentation was not observed. Third, there was no evidence that α-tomatine induced release of cytochrome c from mitochondria into cytosol. It has been reported that the permeability of the mitochondrial membrane is regulated by AIF and that AIFinduced cell death bypasses caspase activation.21 It has also been reported that survivin inhibition promotes cell death independently of cell cycle progression in many types of leukemia cell lines.22 In addition, survivin has been shown to suppress intracellular translocation of AIF to the nucleus from mitochondria that could induce the caspase-independent apoptotic pathway. We therefore examined the possibility of whether α-tomatine could directly down-regulate the survivin protein level that leads to cell death. The results show that the expression of survivin was down-regulated in CT-26 cancer cells treated with α-tomatine for 24 h (88% reduction compared to control), where marked cell death was observed (Figure 5A and Table 1). Similar results were obtained with NF-κB/p65, in which α-tomatine suppressed nuclear translocation of the p65 subunit from the cytosol, resulting in growth inhibition of the α-tomatine-treated cells.23 Figure 5B shows that in normal cells, survivin expression blocks nuclear translocation of AIF from mitochondria, suggesting that AIF intracellular translocation and survivin inhibition were strongly involved in α-tomatine-mediated caspase-independent cell death. We examined the translocation of NF-κB because the survivin gene is known to be a target gene regulated by NFκB.24 Antitumor Effects of α-Tomatine in Vivo. To extract the total cell protein, equal amounts of tissue samples were collected and mixed. Relative expression (R.E.) in the figures is an arbitrary value that shows the expression ratio of target protein compared to expressed β-actin as the control protein. Standard deviation does not have statistical significance for R.E. Mean ± SD values were calculated from the average for 10 mice in each group. These values were derived by calculating means ± SD from three independent (separate) experiments. To examine the antitumor efficacy of α-tomatine in vivo, CT26 cancer cells were transplanted intracutaneously onto the backs of the mice. The mice were divided into two groups: a vehicle (control) and α-tomatine treatment group (5 mg/kg, ip injection per day). Figure 6 and Table 2 show that compared to the control, α-tomatine inhibited tumor growth by about 38%. Tumor inhibition was not associated with changes in body, heart, lung, stomach, or liver weight. However, there was a significant 29% increase in spleen weight. In addition, there

Figure 5. Down-regulation of survivin expression and inhibition of AIF nuclear translocation by α-tomatine treatment on CT-26 mouse colon cancer cells. (A) Total cell lysate from α-tomatine-treated cells was subjected to Western blot analysis using survivin-specific antibodies. βActin was used as an internal control for quantifying the modulation rate of survivin expression. (B) Translocation of AIF and NF-κB/p65 to nuclei was assessed by Western blot analysis on nuclear fraction prepared from α-tomatine-treated cells. Each protein expression level was expressed relative expression (R.E.) value calculated from target protein/β-actin or PCNA protein level. The figures represent three independent experiments.

Figure 6. Effects of α-tomatine on tumor growth in vivo. BALB/c mice were intracutaneously transplanted with CT-26 mouse colon cancer cells (1 × 106 cells, 200 μL) and then subjected to intraperitoneal administration of α-tomatine (5 mg/kg body weight in 200 μL of 0.1% DMSO). After 2 weeks, mice were sacrificed and tumors excised to compare tumor growth patterns between experimental and vehicle-treated control group. Tumor weights are expressed as the mean ± SD (n = 10). Bars not sharing the same letters are significantly different between groups at p < 0.05.

were no associations between tumor regression and caspase-8 and -9 activity increases. However, caspase-3 activity increased in response to α-tomatine administration (Figure 7). Western blot analysis was used to measure the expression of AIF and survivin (Figure 8). Consistent with the observed effects of α-tomatine in the vitro system, the α-tomatine increased the nuclear-localized AIF protein level and decreased the survivin level. These findings indicate that α-tomatine induced cell death in vitro and inhibited the transplanted tumor 1146

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Journal of Agricultural and Food Chemistry Table 2. Body and Organ Weights of Mice Intraperitoneally Administered α-Tomatinea organ wt (g/20 g body wt) sample

final body wt (g)

heart

lung

stomach

liver

spleen

vehicle tomatine 5 mg/kg

35.7 ± 2.1 35.5 ± 2.3

0.18 ± 0.01 0.17 ± 0.01

0.29 ± 0.02 0.28 ± 0.03

0.28 ± 0.01 0.26 ± 0.02

1.10 ± 0.12 1.05 ± 0.19

0.15 ± 0.01b 0.21 ± 0.02a

Values are expressed as the mean ± SD of each mouse group (n = 10). Values not sharing the same letters in a column are significantly different at p < 0.05. Vehicle indicates 0.1% DMSO treatment only. a

Figure 7. Effects of α-tomatine on caspase-3, -8, and -9 activities on tumor tissues from mice intracutaneously transplanted with CT-26 mouse colon cancer cells. α-Tomatine (5 mg/kg body weight) or vehicle was administered via peritoneal route into mice. The enzymatic activities of caspase-3, -8, and -9 in the tumor tissue lysates were determined by incubation with specific colorigenic substrates DEVDpNA, IETD-pNA, and LEHD-pNA, respectively. Enzyme activities are expressed as the mean ± SD (n = 10). Bars not sharing a common letter are significantly different between groups at p < 0.05. The figures represent three independent experiments.

growth in vivo by survivin inhibition and AIF induction, indicating that anticancer effects of α-tomatine might be exerted through caspase-independent pathway in both CT-26 cultured cancer cells and the mice tumor transplanted with the colon cancer cells.

Figure 8. Down-regulation of survivin expression and inhibition of AIF nuclear translocation by α-tomatine treatment on mouse tumors transplanted with CT-26 mouse colon cancer cells. (A) Total tumor tissue lysate from the mice ip administered α-tomatine was subjected to Western blot analysis using survivin-specific antibodies. β-Actin was used as an internal control for quantifying modulation rate of survivin expression. (B) Nuclear translocations of NF-κB/p65 from cytosol and AIF from mitochondria were assessed by Western blot analysis on nuclear fraction prepared from the tumor from the mice administered α-tomatine via peritoneal route. Each protein level was expressed as the R.E. value calculated from target protein/ β-actin or PCNA protein level. The figures represent three independent experiments.

DISCUSSION Anticarcinogenic Effects of α-Tomatine against CT-26 Cells in Vitro and in Vivo. The cited data imply that αtomatine induced apoptosis of CT-26 cells and that cell death occurred through caspase-independent pathways. This suggestion is reinforced by the observed failure of a pan-caspase inhibitor, z-VAD-fmk, to block cell death. A journal reviewer suggested we validate the concentration of z-VA-fmk used in the present study. Table 1 gives the concentration of z-VAD-fmk used. However, we believe that it is not necessary to validate the concentration of z-VAD fmk because this concentration (50−100 μM) was widely used in related earlier studies. We therefore used 100 μM as the working concentration above the saturation point. In addition, increases in the cell viability observed in the control group treated only with z-VAD-fmk might have resulted from suppression of initiation of spontaneous apoptosis. The cited results show that nuclear localization of AIF and inhibition of survivin expression were strongly involved in the α-tomatine-mediated, caspase-independent cell death and that the tumor-growth-inhibiting effect of α-tomatine was not accompanied by adverse effects such as changes in organ weights, with the possible exception of an increase in spleen

weight. In addition, there was no apparent association between tumor regression and increases in caspase-8 and -9 but not caspase-3 activities. We have no obvious explanation for this difference. Western blot analysis used to measure the in vivo expression of AIF and survivin paralleled the observed effects induced by α-tomatine in vitro; α-tomatine increased the AIF level and decreased the survivin level. Taken together, the described findings indicate that α-tomatine inhibited the cancer cells in vitro and tumor growth in vivo by inhibition of survivin and induction of AIF. It seems that these anticarcinogenic effects of α-tomatine appear to be exerted largely through a caspaseindependent pathway in both the CT-26 cultured cancer cells and in the transplanted mouse colon tumor cells. End Points of Cytotoxicity and FACS Assays. These two assays measure different end points. The two cytotoxicity assays used in this study measure cellular dehydrogenase activities as an indicator of cell viability. The FACS analysis measures the incorporation of PI into nuclei through the gap generated by the collapse of the cell membrane together with staining of phosphatidylserine that is localized on the inner side of the lipid bilayer that flipped over to the outer side. The FACS analysis

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Journal of Agricultural and Food Chemistry

• α-Tomatine inhibited human leukemia MOLT-4 cells by activating cell cycle checkpoints without damaging single or double DNA strands in the cells.31 The slowing of the cell cycle seems to involve caspase-independent cell death associated with an increase in the tumor suppressor protein p53, and the pro-apoptotic protein PUMA (p53 up-regulated modulator of apoptosis), as well as other protein biomarkers associated with cell cycle regulation. • α-Tomatine-mediated anticancer activity against human chronic myeloid leukemia cells in vitro and in a transplanted mouse tumor operated through cell-cycle and caspase-independent pathways.32 The mechanism against the leukemia cells seems consistent with that described in the present study for the colon cancer cells. • We found that feeding 2000 ppm of tomatine and 224 ppm of the multiorgan carcinogen dibenzo[a,l]pyrene (DBP) to rainbow trout for 9 months resulted in a reduced incidence of liver and stomach tumors by 41.3 and 36.3%, respectively, as compared to the incidence of tumors observed with DBP alone.33 • Ip-administered α-tomatine (5−10 mg/kg) significantly attenuated the growth of androgen-independent PC-3 prostate cancer cells in mice.34 Tumor-suppression was associated with increased apoptosis and by reduced nuclear translocation of the p50 and p65 gene components of the NF-κB signaling pathway. This study seems to provide evidence that α-tomatine can inhibit the growth of prostate cancer tumors in vivo without inducing overt toxicity. • The activity of α-tomatine generally decreases upon removal of one or more of the four carbohydrate groups of the lycotetraose side chain.35 For example, the activity of α-tomatine against the PC-3 prostate cancer cells was about 200 times greater than that of the aglycone tomatidine. By contrast, tomatidine was highly active against the foodborne pathogen Staphylococcus aureus,36 suggesting that the two bioactivities are governed by different mechanisms. • Ip-injected α-tomatine at 1 mg/kg slowed the growth of Ehlich tumor in mice; the combination of α-tomatine (1 mg/kg) and the cancer drug doxorubicin (2 mg/kg) acted synergistically and extended survival of the mice with the Ehrlich tumors.37 The activity against the tumors, which are derived from adenocarcinoma of the mammary gland, seems to involve the immune system in the inhibition of tumor progression. This suggestion seems plausible on the basis of reports by other investigators that tomatine contributes to the protective immunity of rodents.38,39 The results of the present study on the inhibition of tumor growth in mice with implanted colon cancer complement and extend the cited in vitro and in vivo studies by other investigators. It seems that the mechanistic molecular, signaling, biomarker, and immunomodulating events associated with apoptosis and cell migration might not be the same for the different cancer cell lines. Overall, the cited observations imply that both intrinsic and extrinsic pro-apoptosis pathways are involved and that α-tomatine may protect against development and progression of several cancers (multiorgan protection). This is not surprising because carcinogenesis is a sequential multistage cellular process consisting of tumor initiation, tumor

data show only the population of cells that are susceptible to apoptosis and not directly associated with cell death. By contrast, the MTT assays measure cell death. Reported Anticarcinogenic Activities and Mechanisms of α-Tomatine. To place the findings of the present study in proper perspective, we will briefly mention results of reported studies on the anticarcinogenic potential of αtomatine in vitro and in vivo. • Using a microculture tetrazolium (MTT) in vitro assay, we previously reported that tomatine is a strong inhibitor of growth for both human colon and liver cancer cell lines, as evidenced by the dose-dependent (0.1−100 μg/ mL) inhibition of HT29 colon cancer cells at levels ranging from 38.0 to 81.5% and of human HepG2 cancer cells, from 46.3 to 89.2%.25 The anticarcinogenic activity against human liver cancer cells at a tomatine concentration of 1 μg/mL was higher than the corresponding activity observed with the commercial anticancer drug doxorubicin. • We also investigated six green and three red tomato extracts for their ability to induce cell death in human cancer and normal cells using a microculture MTT assay.26 Compared to untreated controls, the hightomatine green tomato extracts strongly inhibited the following human cancer cell lines: breast (MCF-7), colon (HT-29), gastric (AGS), and hepatoma (liver) (HepG2), as well as normal human liver cells (Chang). • α-Tomatine (a) inhibited cell invasion and migration and phosphorylation of Akt and extracellular signal-regulated kinases 1 and 2 (ERK1/2) in human lung adenocarcinoma A549 cells; (b) did not affect phosphorylation of cJun N-terminal kinase (JNK) and the p38 gene; (c) decreased nuclear levels of NF-κB, c-Fos, and C-Jun; and (d) inhibited binding abilities of NF-κB and activator protein-1 (AP-1).27 Inhibition of metastasis occurs by reducing MMP-2, MMP-9, and urokinase-type-plasminogen activator (uPA) activities through the phosphoinositide 3-kinase/Akt (PI3K/Akt or ERK1/2) signaling pathway and inhibition of NF-κB or AP-1 binding activities. • Another study confirmed that α-tomatine suppressed invasion and migration of human non-small lung cancer NCI-H460 cells through inactivation of the FAK/PI3K/ Akt signaling pathway and lowering binding activity of NF-κB.28 Cytotoxicity occurred through inactivation of the signaling pathway and enhancement of IκBα protein expressions to reduce NF-κB DNA binding activity, resulting in down-regulation of MMP-7 expression, inhibition of cell migration and invasion, and interference with the rearrangement of the actin cytoskeleton by decreasing the expression of the pFAK protein. • α-Tomatine inhibited PC-3 prostate cancer cells with an IC50 value of 1.67 μM.29,30 Cytotoxicity occurred after an hour of treatment and was mainly due to induction of apoptosis as evidenced by decreased mitochondrial membrane potential and increased nuclear condensation, polarization of F-actin potential, cell membrane permeability, cytochrome c expression, induction of activation of caspase-3, -8, and -9, inhibition of NF-κB nuclear translocation, and a decrease in NF-κB/p50 and NFκBp65 in the nuclear fraction. 1148

DOI: 10.1021/jf5040288 J. Agric. Food Chem. 2015, 63, 1142−1150

Article

Journal of Agricultural and Food Chemistry

peritoneal; HRP, horseradish peroxidase; MMP, matrix metalloproteinase; MTT, a tetrazolium dye, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; NF-κB, nuclear factor kappa B; NP-40, Tergitol-type NP-40 detergent; PI, propidium iodide; PCNA, proliferating cell nuclear antigen; PS, phosphatidylserine; PI3K, phosphoinositide 3-kinase; PI3K/ AKT/mTOR, an intracellular signaling pathway; RIPA, radioimmunoprecipitation assay; z-VAD-fmk, pan-caspase inhibitor; XIAP, X-chromosome-linked inhibitor of apoptosis

promotion, and tumor progression characterized by dysregulation of multiple genes resulting in multiple adverse symptoms. Each stage involves signaling pathways and associated biomarkers. Safety of Tomatine. The following reported observations indicate that tomatine does not appear to be toxic to animals or humans. (a) The following LD50 value have been reported for tomatine in mice (in mg/kg body weight): ip, 25− 33.5; iv, 18; oral, 500; subcutaneous, >1000.15,40 (b) As part of the anticarcinogenic study of feeding tomatine to rainbow trout for 9 months, we found that tomatine did not induce changes in mortality, fish weights, liver weights, or tissue morphology.33 We also observed a lack of toxicity in hamsters orally fed tomatine.41 (c) The apparent nontoxicity of tomatine in animal models is reinforced by the fact that Peruvians consume without deleterious effects high-tomatine red tomatoes grown in the Andes mountains8 and that high-tomatine “pickled green” and “green-fried” tomatoes are part of the human diet. (d) The present study shows that except for the spleen, the body and organ weights of the mice were not affected by their exposure to tomatine. Pancreas weights were not determined. Outlook. Because our previous studies found that dietary tomatine and high-tomatine green tomatoes decreased plasma low-density (bad) cholesterol and plasma triglyceride in hamsters fed a diet high in saturated fat and cholesterol by 41 and 47%,41,42 chemopreventive effects of α-tomatine and high-tomatine tomatoes against both cancers and cardiovascular disease in humans merit further study. Such studies should evaluate pure tomatine and high-tomatine green tomato diets. A key consideration in cancer prevention and treatment should be the ratio of effective preventive or therapeutic to toxic dose. Combinations of low doses of tomatine with cancer drugs that might act synergistically with reduced side effects also merit study. There is also a need to develop high-tomatine red tomatoes by suppressing the genes in the tomato plant that govern the biosynthesis of enzymes that degrade most of the tomatine during ripening of tomatoes.43−46 Such new red tomatoes will contain two highly bioactive health-promoting molecules, αtomatine and the red pigment lycopene.40,47,48

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*(S.H.N.) Phone: 82-31-219-2619. Fax: 82-31-219-1615. Email: [email protected]. *(M.F.) Phone: (510) 559-5615. Fax: (510) 559-6162. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED AIF, apoptosis-inducing factor; Akt, protein kinase B; annexin V-FITC, annexin V conjugated with fluorescein isothiocyanate, for detection of cell apoptosis; AP-1, activator protein-1; BSA, bovine serum albumin; Bcl-2, Bcl-xL, anti-apoptotic proteins; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; IC50, concentration of the test substance that inhibited 50% of the cancer cells; IgG, immunoglobulin; ip, intra1149

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DOI: 10.1021/jf5040288 J. Agric. Food Chem. 2015, 63, 1142−1150