Flesh extract of Mangifera indica possesses anti-proliferation effect on different types of cancer cells. However, the effect of its seed kernel extract (MSP) on the growth of human colorectal carcinoma cells (CRC) has not yet been evaluated. Phenolic species of MSP were extracted and measured by colorimetry. Two CRC cell lines (Colo 320DM and SW480) were treated with MSP and assessed for viability by trypan blue exclusion, for cell cycle distribution by flow cytometry, for apoptosis by annexin V labeling, for mitochondria potential by rhodamine 123 staining and for changes in the levels of proteins involved in cell cycle control or apoptosis by immunoblotting. MSP inhibited the proliferation (12.5 μg/mL - 50 μg/mL) of Colo 320DM and SW480. MSP inhibited proliferation by blocking cell cycle progression at G1 (SW480) or S (Colo 320DM) phase and inducing apoptotic death. Western blotting indicated that MSP-blocking cell cycle was associated with cyclin levels. MSP-treated Colo 320DM and SW480 also showed activation of caspase 8, 9 and 3. MSP induces cell cycle arrest and apoptotic death in two CRC cell lines. The results indicate that MSP is a potential novel chemoprevention and treatment agent for colorectal cancer.
Colorectal carcinoma (CRC) is the most common cancer in Taiwan since there were 12,488 newly-diagnosed CRC patients in 2009. CRC is a progressive disease which often begins from benign tumors or inflammatory disorders. After gradual accumulation of gene mutations, chromosomal instability and epigenetic changes, the malignant carcinoma would form and threaten the patients’ life [
Fruit seeds contain many protective phenolic compounds, making them of interest to the nutraceutical industry [
Roswell Park Memorial Institute (RPMI) media 1640, Leibovitz L-15 and Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), L-glutamine, trypsin and antibiotics were purchased from Gibco Ltd. (Paisley, UK). Proteinase inhibitor cocktail, sodium orthovanadate, NaF, sodium pyrophosphate, Triton X-100, ammonia persulfate, N,N,N’,N’-tetramethylethylenediamine (TEMED), Tween 20, rhodamine 123, propidium iodide, gallic acid and catechin were purchased from Sigma (St. Louis, MO). Bicinchoninic acid (BCA) protein assay reagent was from Pierce (Rockford, IL). Acrylamide was purchased from Bio-Rad (Hercules, CA). Polyvinylidene fluoride (PVDF) membrane (Immobilon-P) was purchased from Millipore (Bedford, MA). Mouse monoclonal anti-caspase 3, cyclin A, cyclin D1 and Cyclin E antibodies were purchased from Zymed (San Francisco, CA). Goat polyclonal anti-poly [ADP-ribose] polymerase (PARP), Tp53 and CIP 1/p21 antibodies and goat anti-rabbit, anti-mouse and rabbitanti-goat secondary antibodies conjugated with horseradish peroxidase (HRP) were purchased from R&D Systems (Minneapolis, MN). Annexin V conjugated with FITC was fromGene Research (Taipei, Taiwan).
Human CRC cell lines SW480 and Colo 320DM and rat small intestine epithelial cell IEC-6 were obtained from the Bioresource Collection and Research Center, Taiwan. SW480, established from a primary adenocarcinoma of a colon cancer patient with Duke’s stage B, were cultured in 90% Leibovitz L-15 supplemented with 10% heat-inactivated FBS. Colo 320DM, derived from a moderately differentiated adenocarcinoma of a colon cancer patient, were cultured in 90% RPMI medium 1640 supplemented with 10% heat-inactivated FBS. IEC-6 were cultured in 90% DMEM supplemented with 0.1 Unit/ml bovine insulin and 10% FBS. All media used here were supplemented with 25 U/mL penicillin and 25 μg/mL streptomycin as antibiotics. The cells were incubated at 37˚C in a 95% air/5% CO2 and water-saturated atmosphere, except that SW480 were in room air. All experiments were carried out on cell lines passaged 5 - 20 times. Mango fruit was purchased from a certified Mango farm (Tainan, Taiwan).
Mango fruit was washed and peeled, and then the seeds were dried in a 70˚C oven for at least 48 h. The dried seeds were then ground using a stainless-steel grinder (RT-02, Rong Tsong Iron Factory Incorporation, Taiwan). The powder was sealed and stored at −20˚C, or applied to extraction of the polyphenol compounds. For extraction, Mango seed powder was refluxed with a10 times (v/w; mL/g) ratio of 70% ethanol solution overnight. The crude extract was filtered through No 1 filter paper, and then centrifuged at 3000 rpm for 30 min. The supernatant was concentrated using a rotary evaporator under reduced pressure in a water bath at <35˚C and, then, freeze-dried. The final crude extract was defined as MSP, in which polyphenol species named total phenols, total flavonoids and condensed tannins were measured by colorimetry methods as previously described [
Two CRC cell lines and IEC-6 cells were plated at 100,000 cells in 60-mm tissue culture dishes. After 18 h of culture, cells were treated with different concentrations of DMSO-dissolved MSP (0, 12.5, 25 and 50 μg/mL). At 24 h, cells were collected by trypsinization, stained with trypan blue, and the cell number in suspension was counted in duplicate using a hemocytometer. Data were the average of three independent experiments.
MSP treated cells were harvested by trypsinization and washed with phosphate-buffered saline, then ethanol-fixed in 70% ethanol at −20˚C for at least 30 min. Fixed cells were collected by centrifugation and reconstituted in phosphate-buffered saline. Cells were then stained with propidium iodide solution (20 μg/mL propidium iodide and 10 μg/mL RNase A) at 37˚C in the dark for 30 min. The stained cells were examined by flow cytometry using FL-2A to score DNA content of cells. Percentages of G1, S and G2/M cell cycle phases were determined with Modfit software (Verity Software House, Inc., Topsham, ME).
Apoptosis measurement was carried out using annexin V to label cell surface phosphatidylserine of apoptotic cells [
ΔΨm measurement was carried out essentially as described by Hsu et al. [
MSP-treated cells were washed with ice-cold phosphate-buffered saline and lysed in homogenization buffer (10 mM Tris-HCl at pH 7.4, 2 mM EDTA, 1 mM EGTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1:100 proteinase inhibitor cocktail) on ice for 30 min. After centrifugation for 30 min at 13,000 rpm at 4˚C to remove insoluble materials, the lysate was determined the protein concentration by BCA protein assay kit and then separated on SDS-PAGE. The resolved bands were electrotransferred to PVDF membranes using a semi-dry blot apparatus (Bio-Rad) at 3 mA per cm2 of the gel in transfer buffer (25 mMtris, pH 8.3, 192 mM glycine, and 20% methanol) at room temperature for 30 min. Immunoblotting was performed by incubating PVDF membranes with 5% non-fat milk in Tris-buffered saline supplemented with Tween 20 (TBST, 10 mM Tris, pH 7.4, 150 mM NaCl, 0.2% Tween 20) for 1 h at room temperature for blocking the residue free protein binding sites on PVDF. The membrane was incubated with different primary antibodies in 3% non-fat milk in TBST at 4˚C for 18 h. After repeating washing with TBST, the membrane was incubated with secondary antibodies conjugated with HRP. Immunoblots were developed using enhanced chemiluminescence and the luminescence visualized on a chemoluminescence detection system (Bio-Rad).
All data are expressed as means + standard deviation (SD) unless stated otherwise. Differences between groups were calculated using the Student's unpaired t-test. Dose-dependent effect was calculated using simple linear regression. P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 12.0 (SPSS, Inc., Chicago, IL).
As shown in
To determine the cellular and molecular mechanism of growth inhibition of MSP on CRC cells, we investigated cell cycle progression and the change of the expression levels of cell cycle controlling proteins after MSP treatment. As shown in
Phosphatidylserine translocation was assessed to determine apoptosis of MSP-treated CRC cells by staining with FITC-conjugated annexin V. Annexin V positive cells gradually increased in Colo 320DM cells and SW480 cells after which were treated with more than 12.5 μg/mL MSP (
membrane potential loss, assessed by measurement of rhodamine 123 staining also decreased in MSP-treated Colo 320DM and SW480 cells (
We here investigated MSP growth inhibition in two CRC cell lines. MSP inhibited proliferation of Colo 320DM and SW480cells. To our best knowledge, this is the first evidence that MSP inhibits proliferation of CRC cells. Recent studies show that Mango extract or its important component mangiferin could suppress the growth of lung, colon, prostate, breast cancer cells and leukemia cells [
of Mango named Mangifera pajang possessed the inhibition ability to breast cancer cell proliferation[
Cell cycle arrest and apoptosis are two main cellular mechanisms that polyphenols inhibit the growth of CRC cells [
Apoptosis induction is another possible mechanism of MSP in CRC cells. In the present study, we showed that MSP induced apoptosis significantly in Colo 320DM and SW480 cells at concentrations more than 12.5 μg/mL. MSP-induced apoptosis was mediated by caspase 3activation. Many reports suggest that polyphenolic-induced apoptosis in CRC cells was mediated by activation of caspase 3 and the elevating cleavage fragments of its substrate PARP was a functional marker to imply caspase 3 activity [
MSP-induced caspase 3 activation and apoptosis may operate through caspase 8-mediated apoptosis. Caspase8is one of the cysteine proteases involved in apoptosis. The resting stage of caspase 8 is an inactive single polypeptide chain zymogen procaspase, named pro-caspase 8. When ligand binding-induced trimerization of death receptors occurs, the receptor-specific adapter protein Fas-associated death domain (FADD) is recruited. This protein complex then recruits pro-caspase8and is activated by proteolytic cleavage. Activated caspase 8 then communicates the apoptosis signal either by directly cleaving and activating downstream caspases such as caspase 3 or by cleaving the BH3 Bcl2-interacting protein, which results in increasing mitochondria permeabilityand the release of cytochrome c from mitochondria to cytosol, promoting activation of caspase 9 in a complex with dATP and Apaf-1 [
In conclusion, MSP inhibited CRC cells proliferation mainly through cell cycle arrest at either G1 or S phase, through regulating levels of cyclins. MSP may operate through apoptosis by activating caspase 8 and directly triggers caspase 3 activation or passes through mitochondria membrane potential disruption and the cleavage of pro-caspase 9. We found that MSP treatment possesses anti-proliferative effect in CRC cell lines, suggesting its potential as a novel chemopreventive agent for CRC.
This work was supported by the grants NSC 99-2320-B-415-002-MY3 (C.-H. Chen) and NSC 102-2320-B-415- 005-MY3 (C.-H. Chen) from the National Science Council, Taiwan, ROC.