Stereoisomers of Astaxanthin Inhibit Human Colon Cancer Cell

Article pubs.acs.org/JAFC

Stereoisomers of Astaxanthin Inhibit Human Colon Cancer Cell Growth by Inducing G2/M Cell Cycle Arrest and Apoptosis Xiaojuan Liu,*,†,‡ Mingyue Song,‡ Zili Gao,‡ Xiaokun Cai,‡ William Dixon,‡ Xiaofeng Chen,† Yong Cao,† and Hang Xiao*,‡ †

Department of Food Science, College of Food Science, South China Agricultural University, Guangzhou 510642, China Department of Food Science, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States

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ABSTRACT: Astaxanthin (AST) is a xanthophyll carotenoid with potential protective effects against carcinogenesis. Different stereoisomers of AST (ASTs) exist in a variety of food sources. Due to limited information on the bioactivities of ASTs, the present study investigated the inhibitory effects of ASTs on HCT116 and HT29 human colon cancer cells. ASTs investigated herein included 3S,3′S (S) from Haematococcus pluvialis, 3R,3′R (R) from Phaf f ia rhodozyma, and a statistical mixture (S: meso: R = 1:2:1) (M) from synthetic AST. Cell viability assay showed that ASTs all inhibited colon cancer cell growth in a timedependent (24−72 h) and dose-dependent (4−16 μM) manner, and there was no significant difference among the IC50 values of ASTs (p > 0.05). Flow cytometry analysis indicated that ASTs induced G2/M cell cycle arrest and cellular apoptosis in cancer cells. The cell cycle arrest caused by ASTs was associated with increases in the expression levels of p21Cip1/Waf1, p27, and p53, as well as decreases in the levels of CDK4 and CDK6. Meanwhile, the apoptosis induced by ASTs was confirmed by activation of caspase-3 and PARP in the cancer cells. The results indicated that hydroxyl (OH) at C3 and C3′ of terminal ring structure might not be the major factor that affects the anticancer activity of AST. This study revealed important information on the inhibitory effects of ASTs on human colon cancer cells, which provided a basis for using ASTs as chemopreventive agents for colon cancer. KEYWORDS: astaxanthin, stereoisomer, colon cancer, cell cycle, apoptosis

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INTRODUCTION Despite the increasing efforts on cancer research, cancer remains a main factor of human death around the world. Colon cancer is the second leading cause of cancer mortality in the United States.1 Cancer chemoprevention has been considered as a promising approach to controlling cancer death, especially in colon cancer, due to the slow progression of colorectal adenomatous polyps.2,3 Astaxanthin (AST), a xanthophyll carotenoid, widely exists in many living organisms including yeasts, microalgae, bacteria, salmon, crustacean, trout, and shrimp.4 AST has shown various biological activities including antioxidation, anticardiovascular disease, anti-inflammation, anticancer, and immune-modulation effects in animals and in humans.5−8 To date, no obvious adverse effects of AST consumption in animals and humans have been reported.9 Increasing evidence suggests that AST may be a novel and promising chemopreventive agent for inhibiting the proliferation of colon cancer cells.5,10,11 Previous studies have shown that AST-rich alga Haematococcus pluvialis (H. pluvialis) had an inhibition effect on the growth of human colon cancer cells.12 AST also inhibited colitis-associated colon carcinogenesis by decreasing oxidative stress, alleviating chronic inflammation, and suppressing cell proliferation in mice and rats.11,13−15 AST (3,3′-dihydroxy-β,β′-caroten-4,4′-dione) has two ionone rings held together by a long chain of conjugated double bonds, leading to multiple geometrical isomeric forms (cis and trans). Because of the existence of two stereogenic carbon atoms at the C3 and C3′ positions, there are three stereoisomers for AST: a pair of enantiomers [3R,3′R (R) and 3S,3′S (S) AST) and a meso form (3R,3′S AST). Synthetic AST is a statistical mixture (S: meso: R = 1:2:1) (M) (Figure 1). © XXXX American Chemical Society

Different organisms produce AST in different stereoisomeric forms in nature. H. pluvialis is the most abundant source of AST, and it mainly yields S isomer, while the biosynthesis of AST in yeast Phaf fia rhodozyma (P. rhodozyma) mainly yields R isomer.16,17 Furthermore, S AST is the predominant stereoisomer produced in salmon tissue, while a unique mixture of R/meso/S isomers occurs in prawns and crabs.18 Although cis and trans AST isomers have been studied for their biological properties,19−21 the information on the functional characteristics of stereoisomeric AST has been extremely limited. The antioxidant effects of AST was related to the structure of long conjugated chain, hydroxyl (OH) at C3 and C3′ of terminal ring structure, unsaturated ketone (CO), and α-hydroxy ketones that are composed of hydroxy and keto groups,19 therefore it is reasonable to hypothesize that different stereoisomeric AST may show different anticancer activity. In this study, for the first time, we determined the inhibitory effects of three stereoisomeric AST (S, R, and M) on human HCT116 and HT29 colon cancer cells.

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MATERIALS AND METHODS

Chemicals. Synthetic AST (S: meso: R = 1:2:1) (M) was purchased from Sigma-Aldrich Inc. (St. Louis, MO, U.S.A.) and the purity was 97.5%. H. pluvialis and P. rhodozyma powders were purchased from Jingzhou Natural Astaxanthin Inc. (Jingzhou, China) and Igene Biotechnology, Inc. (Columbia, MD, U.S.A.), respectively. HPLCReceived: August 14, 2016 Revised: October 3, 2016 Accepted: October 4, 2016

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DOI: 10.1021/acs.jafc.6b03636 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Structures of S, meso, and R stereoisomers of AST. Cell Cultures and Treatments. HCT116 and HT29 cells are widely used for cancer research. Human colon cancer cell HCT116 and HT29 were obtained from American Type Cell Collection (ATCC, Manassas, VA, U.S.A.). The cells were cultured with RPMI 1640 media supplemented with 5% heat inactivated FBS, 100 U/mL of penicillin, and 0.1 mg/mL of streptomycin at 37 °C with 5% CO2 and 95% air. Cells were kept subconfluent and media were replaced every 3−4 days. DMSO was used as the vehicle to deliver AST to the cells, and the final concentration of DMSO in all culture media was 0.1% v/ v. Cell Viability Assay. HCT116 (2000 cells/well) and HT29 (3000 cells/well) cells were seeded in 96-well plates. After 24 h, media were changed with 200 μL of complete media by the presence of serial concentrations of different stereoisomeric AST (4, 6, 8, 10, 12, 14, and 16 μM) for 24, 48, and 72 h based on the preliminary experiment. If the concentrations of AST were controlled between 4 and 16 μM, then the inhibitory rates of AST stereoisomers on HCT116 and HT29 cells treated for 24 h, 48 h, and 72 h were about 10%−95%. After specified treatment times, culture media were changed with MTT-containing medium for viability assay as we previously described.24 Flow Cytometric Analysis of Cell Cycle and Apoptosis. HCT116 cells (4 × 104 cells/well) were seeded in 6-well plates. After 24 h, cells were treated with three concentrations of different stereoisomeric AST (8, 10, 12 μM) in 2 mL of serum complete media. After another 48 h, floating cells were harvested, and adherent cells were detached by 0.25% trypsin-EDTA. After washing with 1 mL of ice-cold PBS, the cells were then subjected to cell cycle and apoptosis analysis as we previousely described.24 Cell cycle was examined by a BD LSR II cell analyzer (BD Biosciences, San Jose, CA, U.S.A.) and data were processed using Modfit 3.2 software (Verity Software House, Topsham, ME, U.S.A.). Apoptosis was quantified by annexin V/PI double staining followed by an apoptosis detection kit (Biovision, Mountain View, CA, U.S.A.). Early apoptotic cells and late apoptotic/necrotic cells were identified using a BD LSR II cell analyzer (BD Biosciences, San Jose, CA, U.S.A.).3 Immunoblotting. HCT116 cells were seeded in cell culture dishes (10 cm). After 24 h, cells were treated with different stereoisomeric AST at 10 μM. After another 48 h, cells were washed with ice-cold PBS, and then subject to Western blotting analysis as we previously described.24 Antibodies for p21Cip1/Waf1, p53, p27, cleaved caspase-3, cPARP, EGFR, p-ERK1/2, CDK4, CDK6, p-p38, p-JNK, and β-actin were used for the immunoblotting. Statistical Analysis. All the results were presented as the mean ± standard deviation (SD). Statistical analyses were performed using

grade methanol, acetone, methyl tert-butyl ether, acetonitrile were from Avantor Performance Materials, Inc. (Center Valley, PA, U.S.A.). RPMI 1640 media, fetal bovine serum (FBS), trypsin-EDTA were from Mediatech Inc. (Herndon, VA, U.S.A.). DMSO, penicillin, streptomycin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich (St. Louis, MO, U.S.A.). Antibodies for p21Cip1/Waf1, p53, epidermal growth factor receptor (EGFR), and phosphorylated c-JUN N-teminal kinase (p-JNK) were from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Antibodies for p27, cleaved caspase-3, cleaved poly ADP ribose polymerase (cPARP), phosphorylated of extracellular signal-regulated kinase p44/42 (p-ERK1/2), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), and phosphop-p38 (p-p38) were from Cell Signaling Technology, Inc. (Beverly, MA, U.S.A.). Antibody for βactin was purchased from Sigma−Aldrich (St. Louis, MO, U.S.A.). Isolation and Chiral Identification of AST. S AST and R AST were isolated from H. pluvialis and P. rhodozyma as we previously described.22 In brief, 1 kg of H. pluvialis powder was extracted by a novel continuous phase transition extraction unit using n-butane as the solvent at 0.55−0.60 MPa and 45 °C for 2 h. R AST was extracted using an ultrasonic extraction method with acetone. The S AST extract was saponified with 0.1 M of KOHCH3OH at 25 °C for 60 min. The S and R AST were isolated by silica gel chromatography, and subsequent recrystallization afforded AST with purity greater than 95%. The purity of S and R AST were tested by HPLC. Analytical HPLC consisted of a pump (LC-15C), a Prominence SPD-M10A diode array detector performing the wavelength scanning from 200 to 400 nm, and an S Shimadzu class LC-VP HPLC system with class LC-VP software (Shimadzu, Tokyo, Japan). The samples were analyzed with a Diamonsil C18 column (250 mm × 4.6 mm i.d., 5 μm, DIKAM, Beijing, China). The mobile phase was methanol−water (9:1, v/v, A) and acetone (B) at a flow rate of 1.0 mL/min. A linear gradient method was used and consisted of 20%−80% acetone for 25 min, 80% acetone for 10 min, and 80%−20% acetone for 5 min. The ambient temperature was controlled at 25 °C. The monitoring wavelength was 476 nm. Chirality was determined by HPLC using a chiralpak IC column composed of cellulose tris (3,5-dichlorophenyl carbamate) polymer. The samples were analyzed by a CHIRALPAK IC chiral column (250 × 4.6 mm2 i.d., 5 μm, Daicel, Osaka, Japan). The mobile phase was methyl tert-butyl ether-acetonitrile (35:65, v/v) at a flow rate of 1.0 mL/min. The detection ambient temperature was controlled at 25 °C, and the monitoring wavelength was 476 nm.23 B


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Figure 2. HPLC (A, B) and HPLC-chiralpak IC (C) analysis of the purified AST and synthetic AST. S AST and R AST were isolated from H. pluvialis and P. rhodozyma, respectively. The purity of AST was determined by a Diamonsil C18 column, and the chirality of AST was determined by HPLC using a CHIRALPAK IC chiral column, as described in the Materials and Methods. SPSS software (SPSS Inc., Chicago, IL, U.S.A.). An analysis of variance (ANOVA) and a post hoc Tukey test with a confidence level of 95% were used to determine the differences among the treatments.

and mainly yield S and R AST, respectively. Therefore, these two materials were used to acquire S and R AST for the following experiments. After a series of preparation processes including extraction, saponification, silica gel column chromatography and crystallization, the purity of S and R AST was 96.16% and 95.69% by HPLC analysis, respectively (Figure 2A,B). On the basis of the chromatogram from HPLC analysis

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RESULTS AND DISCUSSION Purity and Chiral Identification of S, R, and M AST. H. pluvialis and P. rhodozyma are two main sources of natural AST, C


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Figure 3. Growth inhibitory effects of different stereoisomers of AST on HCT116 and HT29 human colon cancer cells. Cancer cells were seeded in 96-well plates. After 24 h, HCT116 cells (A) and HT29 cells (C) were treated with serial concentrations (4 μM-16 μM) of M AST for 24 h, 48 and 72 h. HCT116 cells (B) and HT29 cells (D) were treated with 10 μM of S, R, and M AST for 24 h, 48 and 72 h. After desired treatment times, cell viability was measured by MTT assay as described in the Materials and Methods. Data are expressed as the mean ± SD. The different letters in the column denoted values that were significantly different in different concentrations (A) and different treatment times (B) (p < 0.05, n = 6).

with chiralpak IC column, the chiral structures of AST from H. pluvialis and P. rhodozyma were almost S and R stereoisomers, respectively, while synthetic AST is a statistical mixture (S: meso: R = 1:2:1) (Figure 2C). The results were in agreement with previous reports stating that H. pluvialis and P. rhodozyma were good sources for S and R AST respectively, while synthetic AST is a statistical mixture.23,25,26 S, R and M AST Inhibited the Growth of Human Colon Cancer Cells. In order to determine the anticancer potential of different stereoisomers of AST, we studied effects of S, R, and M AST isomers on the growth of HCT116 and HT29 human colon cancer cells using a cell viability (MTT) test. The results showed that all three stereoisomeric AST showed growth inhibition on cancer cells in a dose-dependent and timedependent fashion. The inhibitory rate (IR) of each stereoisomer was enhanced dramatically with increased AST

concentration and the extension of treatment time. For example, the IR of M AST increased from 10%−18% at 4 μM to 78%−95% at 16 μM after 72 h of treatment (Figure 3A and 3C). Similar trends were also obtained in HCT116 and HT29 cells after S and R treatments (data not shown). At 10 μM, the IR of three stereoisomeric AST on HCT116 cells increased from 35%−39% after 24 h of treatment to 63%−66% after 72 h of treatment (Figure 3B). There was no significant difference in the IR at the same doses and the same treatment times among the three stereoisomers (Figure 3B and 3D). For example, the IC50 values of AST after 72 h of treatment were 7.42, 7.48, and 7.51 μM in HCT116 cells for S, R, and M AST, respectively. Therefore, our data demonstrated that different configurations of AST acted as a growth-inhibitory agent against colon cancer cells with similar potency. Inhibitory effects of AST in various tumor cells have been reported, D


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Figure 4. Cell cycle distribution of HCT116 human colon cancer cell after treatments with different stereoisomers of AST. HCT116 cells were seeded in 6-well plates, and after 24 h, cells were treated with 0 (A), 8 μM (B), 10 μM (C), and 12 μM (D) of M AST, 12 μM of S AST (E), and 12 μM of R AST (F). After another 48 h, cells were harvested and subject to cell cycle analyses as described in the Materials and Methods. The cell population at G0/G1, S, and G2/M phases after treatments with different concentrations of M AST (G) or different stereoisomers of AST (H) were analyzed. All data represent mean ± SD, and different notations in the bar charts indicate statistical significance (p < 0.05, n = 3).

including colon,15 mouth,27 breast,28 gastric,29 and liver tumor cells.30 However, no study has been reported on the

comparison of anticancer effects among different stereoisomers of AST. For the first time, our results showed that the different E


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Figure 5. continued

F


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Figure 5. Effects of different stereoisomers of AST on apoptosis of HCT116 colon cancer cells. HCT116 cell were seeded in 6-well plates, and after 24 h, cells were treated with 0 (A), 8 μM (B), 10 μM (C) and 12 μM (D) of M AST, 12 μM of S AST (E), and 12 μM of R AST (F). After another 48 h, cells were harvested and subject to annexin V/PI costaining assay as described in the Materials and Methods. Quantification of early apoptosis (Q4) and late apoptosis (Q2) resulting from different concentrations of M AST (G), and different stereoisomers of AST (H) were shown. All data represent mean ± SD, and different notations in the bar charts indicate statistical significance (p < 0.05, n = 3).

was increased, which suggested that the continuity and coordination between the different stages of the normal cell cycle had been blocked to result in a G2/M phase cell cycle arrest. S, R, and M AST Induced Cellular Apoptosis in HCT116 Cells. As a programmed cell death process, apoptosis is a mechanism to remove unwanted or damaged cells from the organism. Deregulation of apoptosis can destroy the balance between cell proliferation and cell death and can potentially promote carcinogenesis. Therefore, the induction of apoptosis in precancerous and cancer cells is a valid approach for cancer prevention and treatment.32 To determine the extent to which apoptosis responsible for the growth inhibition caused by three stereoisomeric AST, we determined the effects of AST on cellular apoptosis of HCT116 cells. As shown in Figure 5, three stereoisomers of AST all induced extensive apoptosis in HCT116 cells in a dose-dependent manner. For example, M AST at 8 μM, 10 μM and 12 μM increased early apoptotic cell population by 38.25-fold (21.8%), 46.67-fold (26.60%), and 52.75-fold (30.07%) compared to that of the control (0.57%), respectively. Moreover, M AST at 8 μM, 10 μM and 12 μM also increased late apoptotic cell population by 19.21-fold (9.03%), 25.79-fold (12.12%) and 31.91-fold (15.00%) compared to that of the control (0.47%), respectively (Figure 5G). Similar trends were also examined in HCT116 cells after treatments with S or R AST (Data not shown). There were no significant differences among the different stereoisomers of AST on cellular apoptosis of HCT116 cells. For example, at 12 μM, the apoptotic cell population induced by S, R and M AST was 30.27%, 28.07%, and 30.07% for early apoptosis and 16.42%, 18.20%, and 15.00% for late apoptosis, respectively (Figure 5H). Deregulation of cell cycle progression and apoptosis lead to uncontrolled cell proliferation, which is the symbol of carcinogenesis.33,34 Thus, one of the potentially effective methods for cancer chemoprevention and treatment is inducing cell cycle arrest and apoptosis in cancer cells. The results of flow cytometry analysis obviously showed that S, R and M AST treatments not only caused significant cell cycle arrest at G2/M phase, but also led to extensive cellular

configurations of AST did not lead to significant difference in the grow-inhibitory effects on HCT116 and HT29 colon cancer cells. This might be due to (i) the configuration of stereogenic carbon atoms at the C-3 and C-3′ positions contributed little to the overall anticancer effects, and/or (ii) the polyene chain was the major factor responsible for anticancer effects. In agreement with our results, it has been reported that the individual stereoisomers (S, R, and meso isomers) and the statistical mixture of stereoisomers (M) of AST showed no significant differences in their scavenging efficiency on superoxide, suggesting that the polyene chain might be the main structure responsible for superoxide scavenging.31 S, R, and M AST Caused G2/M Cell Cycle Arrest in HCT116 Cells. To elucidate the mechanism contributed to the inhibition of cell growth by S, R, and M AST in HCT116 colon cancer cells, the effects of these stereoisomers on the cell cycle of cancer cells were determined. As shown in Figure 4, in the absence of AST, abundant HCT116 cells (about 50%) were in G1/S phase due to their high proliferative status (Figure 4A). However, the three stereoisomeric AST all caused significant, dose-dependent increase in the percentage of G2/M cell population after 48 h treatment (Figure 4B−F).Treatments with M AST in HCT116 cells increased the percentage of cells in G2/M phase to 1.37-, 1.86-, and 2.36-fold of the control cells at 8, 10, and 12 μM (p < 0.05) (Figure 4G). S and R AST showed a similar trend as the M AST in modulating cell cycle progression of HCT116 cells (Data not shown). The accumulation of cells in G2/M phase was accompanied by a corresponding decrease in the percentage of cells in G1/S phase. However, there was no significant difference among different configurations of AST in increasing the percentage of cells in G2/M phase of HCT116 cells. For example, at 12 μM, the cell population in G2/M phase treated with S, R, and M AST was 47.28%, 44.85%, and 47.08%, respectively, which were not statistically different from each other, but all significantly higher than that of the control (21.02%) (Figure 4H). Our data suggested that the growth inhibition of HCT116 cancer cells by different stereoisomers of AST was at least partly caused by G2/M cell cycle arrest. The percentage of cells in G2/M phase G


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Journal of Agricultural and Food Chemistry apoptosis in human colon cancer cell HCT116. These findings suggested that the three stereoisomeric AST demonstrated the growth-inhibitory effects on cancer cells by impeding cell cycle progression and increasing programmed cell death. S, R, and M AST Modulated Key Proteins Related to Cell Proliferation, Apoptosis, And Cell Cycle. In order to further clarify the molecular mechanisms underlying the growth-inhibitory effects of three stereoisomeric AST on human colon cancer cell HCT116, we determined the effects of S, R, and M AST on key protein markers associated with cell proliferation, apoptosis, and cell cycle. Immunoblotting analysis indicated that three AST stereoisomers caused extensive changes in these signaling proteins (Figure 6). On the whole, in HCT116 cells, S, R, and M AST at 10 μM significantly

increased expression levels of p21Cip1/Waf1, P27, p53, cleaved caspase-3, cleaved PARP, p-p38, p-JNK, and p-ERK1/2, and decreased expression levels of CDK4, CDK6, and EGFR (Figure 6). p21Cip1/Waf1 and p27, cyclin dependent kinase (CDK) inhibitors, are negative regulators of cell cycle progression. These regulators inhibit CDK activities, and conversely, the inhibition results in decrease cell proliferation.35 After being treated with S, R, and M AST, the protein level of p21Cip1/Waf1 was significantly elevated by 2.7-, 2.4-, and 2.2-fold compared to the control, respectively. S, R, and M AST resulted in a similar effect on the expression level of p27, which was accompanied by the decrease in the level of CDK4 and CDK6. p53 has a variety of cellular functions and plays important roles in inhibiting carcinogenesis. Our results demonstrated that S, R, and M AST treatments significantly increased expression levels of p53 by 1.6-, 2.3-, and 1.5-fold compared to the control, respectively. Enhanced levels of p53 can lead to G2/M cell cycle arrest by downregulating transcription of cyclin B1 and Cdc2.36−38 Furthermore, p21Cip1/Waf1 is one of the transcriptional targets of p53, and increased levels of p53 can lead to increased levels of p21Cip1/Waf1, which in turn causes cell cycle arrest.36,39 Cellular apoptosis of HCT 116 caused by AST stereoisomers was evidenced by the increased levels of cleavage of key apoptosis-related proteins, e.g., caspase-3 and PARP. In HCT116 cells, S, R, and M AST at 10 μM increased the levels of cleaved caspase-3 by 3.7-, 3.5-, and 3.4-fold compared to the control, respectively. Moreover, S, R, and M AST at 10 μM elevated the level of cleaved PARP by 2.0-, 2.4-, and 2.8-fold compared to the control, respectively. Caspase-3 plays an important role in both death receptor-mediated and mitochondria-mediated apoptosis. The cleavage of caspase-3 activates the protein and leads to proteolytic cleavage of downstream proteins, such as PARP. PARP is the key player in DNA repair, the inactivation (cleavage) of PARP by caspase-3 leads to the accumulation of unrepaired DNA, and finally results in cell death.40−42 As a complicated disease, colon carcinogenesis relates to various genetic and epigenetic events in cell proliferation, cell survival, and tumor angiogenesis.43 The high epidermal growth factor receptor (EGFR) expression is mostly common in colon cancer.44,45 Tumors with EGFR overexpression generally have poor prognosis,46 which have resulted in the clinical usage of EGFR inhibitors to treat colon cancer. Our results revealed that treatments of S, R, and M AST significantly decreased the levels of EGFR in colon cancer cells. A sustained expression of the EGFR has been demonstrated to play an import role in the development of carcinogenesis and in apoptosis induction.47 In addition, JNK, p38, and ERK are three kinases that have been proved to regulate apoptosis, especially in oxidative stressinduced apoptotic cell death.48,49 The results showed that S, R, and M AST induced a marked increase in the level of p-p38, pJNK, and p-ERK1/2, suggesting a role of these protein in the pro-apoptotic effect of AST. These results are in accordance with the previous reports that AST-rich algae H. pluvialis could induce these MAP kinases.12 In conclusion, our results demonstrated the potent inhibitory effects of different stereoisomeric AST on human colon cancer HCT116 and HT29 cells for the first time. The inhibition on the cancer cells were associated with extensive cell cycle arrest and apoptosis caused by S, R, and M AST as the result of modulation of oncogenic signaling proteins. Our results

Figure 6. Effects of different stereoisomers of AST on key proteins related to cell proliferation, cell cycle, and apoptosis in HCT116 human colon cells. Cells were seeded into 10 cm culture dishes for 24 h, and then treated with S, R, or M AST at 10 μM. After another 48 h of incubation, cells were collected for Western blotting as described in the Materials and Methods. The numbers underneath the blots represent band intensity (normalized to β-actin, means of three independent experiments) measured by Odyssey system software. The SDs (all within ±15% of the means) were not shown. The experiments were repeated three times. β-actin served as an equal loading control. * indicates the statistical significance in comparison with the control (p < 0.05, n = 3). H


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indicated that hydroxyl (OH) at C3 and C3′ of terminal ring structure might not be the major factor that affect the anticancer activity of AST, which was not in agreement with our original hypothesis that the anticancer activity of AST was related with the above structures. It is possible that the anticancer effects of AST stereoisomers might be associated with the structure of long conjugated chain, unsaturated ketone (CO), and α-hydroxy ketones. However, our data are yet too preliminary to predict an exact structure−activity relationship. Nevertheless, our results warrant future investigations on the in vivo cancer chemoprevention effects of AST stereoisomers.

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AUTHOR INFORMATION

Corresponding Authors

*Telephone/Fax: +86 2085286234. E-mail: [email protected] (X.L.). *Telephone: +1 (413)5452281. Fax: +1 (413)5451262. E-mail: [email protected] (H.X.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the China Scholarship Council for the scholarship. This research was supported by the National Natural Science Foundation of the People’s Republic of China (31401481).

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ABBREVIATIONS AST, astaxanthin; H. pluvialis, Haematococcus pluvialis; P. rhodozyma, Phaf f ia rhodozyma; S, (3S,3′S); R, (3R,3′R); M, mixture (S: meso: R = 1:2:1); DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IR, inhibitory rate; CDK, cyclin dependent kinase; EGFR, epidermal growth factor receptor; JNK, c-JUN Nteminal kinase; ERK, extracellular signal-regulated kinase; PARP, poly ADP ribose polymerase; ERK1/2, extracellular signal-regulated kinase p44/42

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Stereoisomers of Astaxanthin Inhibit Human Colon Cancer Cell

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