Crocetin Induces Cytotoxicity in Colon Cancer Cells Via p 53-independent Mechanisms

Cancer continues to represent the largest cause of mortality in the world and claims over 6 million lives each year (Abdullaev et al., 2000). An extremely promising strategy for cancer prevention today is chemoprevention, which is defined as the use of synthetic or natural agents (alone or in combination) to block the development of cancer in human beings (Abdullaev et al., 2004).It is a hotspot for cancer chemopreventive drug research to look for safe and effective antitumor drugs from plants, vegetables, herbs. Saffron is a naturally derived plant product from the dried stigma of the Crocus sativus flower (family Iridaceae) that may have biologically useful properties. In fact, saffron extract and its biologically active compounds, including crocin, crocetin, carotene, and safranal, have been shown both in vitro and in vivo to possess antioxidant, anticancer, anti-inflammatory, and memoryimproving properties (Abdullaev et al., 2002; Abdullaev et al., 2004; Das et al., 2010). Crocetin (8, 8-diapo-8, 8-carotenoic acid), which is the major ingredient of saffron that is responsible for its coloring property, is a lowmolecular-weight carotenoid compound characterized by a diterpenic and symmetrical structure with seven double bonds and four methyl groups. This compound exhibits antioxidant (Kanakis et al., 2009; Ordoudi et al ., 2009),


Introduction
Cancer continues to represent the largest cause of mortality in the world and claims over 6 million lives each year (Abdullaev et al., 2000).An extremely promising strategy for cancer prevention today is chemoprevention, which is defined as the use of synthetic or natural agents (alone or in combination) to block the development of cancer in human beings (Abdullaev et al., 2004).It is a hotspot for cancer chemopreventive drug research to look for safe and effective antitumor drugs from plants, vegetables, herbs.
Saffron is a naturally derived plant product from the dried stigma of the Crocus sativus flower (family Iridaceae) that may have biologically useful properties.In fact, saffron extract and its biologically active compounds, including crocin, crocetin, carotene, and safranal, have been shown both in vitro and in vivo to possess antioxidant, anticancer, anti-inflammatory, and memoryimproving properties (Abdullaev et al., 2002;Abdullaev et al., 2004;Das et al., 2010).Crocetin (8,, which is the major ingredient of saffron that is responsible for its coloring property, is a lowmolecular-weight carotenoid compound characterized by a diterpenic and symmetrical structure with seven double bonds and four methyl groups.This compound exhibits antioxidant (Kanakis et al., 2009;Ordoudi et al ., 2009),
In order to understand the mechanisms of chemopreventive action of saffron, this study used a p53 gene mutation of Sw480 cells to study the mechanism of the anticancer action of saffron by evaluating its antioxidant, proapoptotic, antiproliferative effects.

Cell culture and Treatment
SW480, NIH3T3 cell lines were obtained from the American Type Culture Collection and cells were cultured in Dulbecco's modified Eagle's medium (DMEM;Hyclone, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) at 37 °C in 5% CO 2 .Cells were plated on 35mm dishes and cultured in DMEM containing 10% FBS until the cells had reached 70% confluence.The crocintreated Cells were incubated with several concentrations of crocetin and the control cells were treated with PBS.

MTT Cell Proliferation and Cytotoxicity Assays
The MTS an improved tetrazolium cell proliferation assay reagent was used in the SW480 cell line to assess the effect on cell proliferation of a range of concentrations of crocin.Cells (5×10 3 ) were plated and grown in 200uL of growth medium in 96-well microtiter plates.After an overnight attachment period, cells were treated with varying concentrations of crocin (0.2, 0.4, 0.8 mmol/L) prepared from a 50 mmol/L stock solution dissolved in ddH2O for 48h.The crocin-treated cells were incubated with MTS at 37 ℃ in a humidified 5% CO 2 atmosphere for 2 hours and product was measured at 570 nm in a microplate reader (Bio-Rad).

Cell-Cycle Analysis by Flow Cytometry
The crocin-treated cells in 0.8 mmol/L were harvested at 6, 24, 48 hours, washed with 4 °C PBS, fixed in 70% ethanol and stored at 4 °C over-night, cells were then treated with RNase A solution (500 U/ml) at 37 °C for 15 min and stained with propidium iodide (PI, 50 μg/ml) at room temperature.DNA content was determined by flow cytometry (Flow CytoMeter, Millipore, USA) using CytoSoft 5.3 software (Millipore).The fractions of cells in G0/G1, S and G2/M phases were determined using FCS Express (version 3, Denovo, USA).

TUNEL Assay
Cells were cultured on 6 well plates with glass slice until the cells had reached 70% confluence, and cells were treated with 0.8 mmol/L crocin.After 6, 24, 48 hours, the crocin -treated cell grown on slices were fixed with 4% paraformalde-hyde and were pre-incubated in 0.1% Triton X-100 (0.01M PBS).Then the slices were incubated with TUNEL reaction mixture (Roche; Switzerland ) for 60 min at 37 °C in a humidified atmosphere in the dark.
The slices were rinsed 3 times with PBS, coverslipped using VectaSHIELD mounting medium containing DAPI (Vector Laboratories, catalog no.H-1200), and analyzed by laser scanning confocal microscope (Olympus FV1000 Viewer).Excitation wave lengths were in the range of 450-500 nm and detection wave lengths in the range of 515-565 nm.

DNA Damage Assay
DNA damage was assessed using the alkaline singlecell gel electrophoresis assay (comet assay).After the crocetin treatments mentioned above, cells were washed in PBS, then incubated in normal medium for 3 h.Cells were then harvested and mixed with low-melting agarose and fixed onto slides.The cells were then lysed and DNA was denatured using an alkaline (pH >14) solution at 4 ℃.Slides were then placed on a horizontal electrophoresis device which allowed damaged and/or broken DNA to migrate away from the nucleus.Slides were then stained with ethidium bromide, visualized by fluorescence microscopy, and analyzed using the Komet 5comet assay analysis software which quantitatively determines the extent of DNA damage in each sample using the mean Olive tail moment calculation as previously described.

Western Blotting
The crocin-treated cell groups (treated with 0.8 mmol/L crocin) and the control cell groups (treated with PBS) were harvested after 48 hours and cells were lysed with cold RIPA lysis buffer containing protease inhibitors, and proteins were collected by centrifugation.Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA) Immunoblots were incubated with the following antibodies: p21 WAF1/Cip1 , P53, cdk2, CyclinA and β-actin.Blots were developed using Supersignal WestPico chemiluminescent substrate (Pierce), imaged and analyzed by photodensitometry with an imaging system (Bio-Rad).

Statistical Analysis
SPSS statistical program (SPSS Inc., Chicago, IL) was used to carry out a one-way analysis of variance (ANOVA) on our data.When significant differences by ANOVA were detected, analysis of differences between the means of the treated and control groups were performed by using Dunnett's t test.

Crocin Induces Growth Arrest and Apoptosis
To test the effect of crocin on cell growth, we carried out the MTS assay.The MTS test showed that crocin significantly reduced the viability of SW480 cells in a dose-dependent manner (Figure 1).For further studies,a crocin concentration of 0.8 mmol/L was used.The effect of crocin on cell cycle progression was also assessed using flow cytometric analysis.crocin-treated SW480 cells displayed an accumulation of the cell population at the S phase starting from 6 hours and in a time-dependent manner (Figure 2).These findings were further supported by measuring the apoptotic cell of green fluorescence after crocin treatment using Tunel assay.Crocin induced apoptosis in SW480 as early as 6 hours after treatment (Figure 3).The apoptosis induction further increased in a time dependent manner reaching 60.14% after 48 hours.The comet assay analysis indicated cells treated with 0.8mmol/L crocin had significantly longer mean tail moments starting from 6 hours (Figure 4) and in a timedependent manner.

Effect of crocin on on the expression of apoptosis-related genes
We choose the cells treated in 0.8 mmol/L and 48 hours to study the mechanism underlying the tumor suppressive effects of crocin.After protein normalization to actin levels, western blot analysis also demonstrated that the level of P53 in crocin-treated SW480 cells no significant difference between crocin-treated NIH3T3, PBS-treated SW480, PBS-treated NIH3T3, normal SW480, normal NIH3T3; there was an apparent decrease in the levels of cdk2, CyclinA protein in crocin-treated SW480 cells, compared with other cells; the level of p21 protein was upregulated, as shown in Figure 5.

Discussion
Natural products have long been used to prevent and treat diseases including cancers and might be good candidates for the development of anti-cancer drugs (Liu et al., 2004).Saffron, a commonly-used spice and food additive, is known for its anti-cancer and anti-tumor properties (Mousavi et al., 2009).Altough most of the in vivo studies were interested in the isolated bioactive compounds of saffron, little research has been done to examine anti-cancer mechanisms of saffron.Crocetin which is the major ingredient of saffron, had been certified two distinct anticancer functions of crocetin as follows: to inhibit cell proliferation at early time by inducing cell cycle arrest in different human cancer cell types via p53dependent and -independent P21 mediated mechanisms and to kill cancer cells via apoptosis et al., 2011).Because p53 is mutated in approximately 50% of human tumors and p53 gene is involved in cell cycle regulation, controlling DNA and apoptosis (Aizat et al., 2011;Heah et al., 2011), we selected SW480 human colonic cancer cell lines with mutation of p53 to study the anticancer effects of crocetin.
The present results were in agreement with previous reports indicating that saffron and its ingredients possessed anti-tumor and anti-cancerogenic activities and have no cytotoxic effect on non-malignant cells (Aung et al., 2007;Tavakkol et al., 2008;Yanatatsaneeji et al., 2010;Harrison et al., 2011).
The results show that crocetin inhibits cell proliferation by inducing S arrest.The proportion of SW480 cells in the S phase at 48 h after crocetin treatment (46.3%) was significantly higher than at 24 h after treatment (P = 0.003), and significantly higher than at 6 h after treatment (p=0.001).No such association between crocetin treatment and proportion of cells in S phase was seen in NIH3T3 cells.These results also show an association between DNA damage and apoptosis level following crocetin treatment, the proportion of apoptotic cells at 48 h after crocetin treatment, when had a significantly longest mean tail moment, was significantly higher than at 24 h (P =0.027) or 6h (P =0.012) after treatment.Consistent with our previous study on SW480 cells, Crocetin induced a significant toxicity start from 6 h and the toxicity seemed to grow stronged over time.The observed apoptotic induction in SW480 cells obviously resulted from DNA damage as reflected by comet assay, suggesting an additional role of Crocetin in sensitizing cancer cells to the effects of other chemotherapeutics.
Progression through the cell cycle is monitored by cell cycle checkpoints that ensure proper replication and segregation of genetic material between daughter cells (Elledge et al., 1996).The eukaryotic cell cycle is controlled by different cyclins and their associated kinases.In mammalian cells, levels of CyclinA and its associated kinase, cdk2, rise in late G1/early S-phase when DNA replication is initiated .The high level of CyclinA may accelerate the progress of S/G2 stage.Without synthesis of CyclinA prior to the S/G2 transition, the cell cannot enter mitosis and the cell cycle will arrest at S phase.In present results , the expression levels of P21 protein increased significantly in the SW480 cells treated with crocetin for 48h, but not in SW480 cells treated with PBS or normal.In contrast, there was an apparent decrease in the levels of CyclinA and cdk2 protein in SW480 cells treated with crocetin for 48h, compared with treated with PBS or normal, whereas there was no significant difference in the level of p53 protein between SW480 cells treated with crocetin and the control cells.
P21, the product of the WAF1/CIP1/SDI1 gene, is an inhibitor of cyclin-dependent kinases and is activated through p53-dependent or p53-independent pathways and it has been reported that P21may contribute to G1 arrest in crocetin-treated cancer cells via inhibits the activity of cyclin dependent kinases (Cdks) or proliferating cell nuclear antigen (PCNA) (Nakano et al., 1997;Lee et al., 1998).Based on these results, it is likely that crocetin decreases CDK2 and cyclins levels in the cells that are sensitive to crocetin, which contributes to increased p21accumulation and crocetin activates p21 through a p53-independent mechanism which could be the one of the main mechanisms of antitumour.
Tavakkol-Afshari and colleagues (Tavakkol et al., 2008) reported that 96% ethanol saffron extract is selectively cytotoxic against epithelial-like human hepatocellular carcinoma cell125s (HepG-2) as well as human cervical carcinoma cells (HeLa) but nontoxic towards normal mouse fibroblast cells (L929).There were the three major carotenoids that are derived from saffron (crocin, crocetin, and dimethylcrocetin) and the additive and synergistic effects among them may enhance its anticarcinogenic properties (Aung et al., 2007).Amr Amin et also confirmed these findings that HepG2 cells treated with the saffron extract contained crocin and safranal increased cleavage of caspase-3, as well as DNA damage and cell cycle arrest (Amin et al., 2011).
Taken together, we suggest three major carotenoids of saffron having the anti-tumor activity but serving different mechanisms and may have synergism anti-tumor activity to some extent.

Figure 1 .
Figure 1.Growth-inhibitory Effect of Crocin on Cells Detected by MTT Assay.Cells were untreated (a) or treated with 0.2 mmol/L(b), 0.4 mmol/L(c), 0.8 mmol/L(d); Absorbance at 490 nm showed a significant decrease in the growth of crocin treated cells compared with control cells (* means P < 0.001 , # means P < 0.05)