Improvement of cytotoxic and apoptogenic properties of crocin in cancer cell lines by its nanoliposomal form

Abstract

Objective: Saffron Crocus sativus L. (Iridaceae) is known for anticancer properties. However, limited effort has been made to correlate these effects to the active ingredients of saffron. In the present study, cytotoxic effects of crocin, the major coloring compound in saffron, and its nanoliposomal form for better cellular delivery are investigated.

Methods: HeLa and MCF-7 cells were cultured and exposed to crocin (1, 2, and 4 mM) and liposomal crocin (0.5 and 1 mM). The 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to assess cytotoxicity. Apoptotic cells were determined using propidium iodide (PI) staining of DNA fragmentation by flow cytometry.

Results: MTT assay revealed a remarkable and concentration-dependent cytotoxic effect of crocin on HeLa and MCF-7 cells in comparison with non-malignant cell line (L929). Crocin liposomal forms (IC₅₀ values after 48 h: 0.61, 0.64, and 1.2 mM) showed enhanced cytotoxic effect compared with the crocin (IC₅₀ after 48 h: 1.603 mM) in HeLa cells. Crocin and its liposomal form induced a sub-G1 peak in flow cytometry histogram of treated cells indicating apoptosis is involved in this toxicity. Liposomal encapsulation enhances apoptogenic effects of crocin on cancerous cells.

Conclusion: It might be concluded that crocin and its liposomes could cause cell death in HeLa and MCF-7 cells, in which liposomal encapsulation improved cytotoxic effects. They could be also considered as a promising chemotherapeutic agent in cancer treatment in future.

Keywords::

• Apoptosis
• crocin
• cytotoxicity
• nanoliposome
• saffron

Introduction

Saffron is the dry stigmas of the plant Crocus sativus L. (Iridaceae) (Abdullaev, 1993). Although it is currently used as a spice and food colorant, traditional medicines have used saffron in the treatment of numerous diseases including cough, colic, insomnia, chronic uterine hemorrhage, cardiovascular disorders, and tumors (Abdullaev and Espinosa-Aguirre, 2004). Crocin is a constituent of C. sativus, which is responsible for the color of the saffron. It has been reported that crocin has different properties including antiplatelet (Liakopoulou-Kyriakides and Skubas, 1990), neuron-protecting (Abe and Saito, 2000), antiatherogenic (He et al., 2005), antioxidant (Chen et al., 2008), aphrodisiac properties (Hosseinzadeh et al., 2008), hypolipidemic effect by inhibiting pancreatic lipase, leading to the malabsorption of fat and cholesterol (Sheng et al., 2006), and anxiolytic-like effect in the rat (Pitsikas et al., 2008). This natural carotenoid protects the brain against excessive oxidative stress and constitutes a potential therapeutic candidate in transient global cerebral ischemia (Zheng et al., 2007). Several studies have showed that crocin has anticarcinogenic and antitumor activities (Fernndez, 2006; Zhao et al., 2008). Crocin possesses significant antiproliferation effects on human colorectal cancer cells (Aung et al., 2007). This carotenoid can induce the significant alteration of gene expression profile of T24 (transitional cell carcinoma of bladder) cell. It is suggested that the antitumor effects of crocin are medicated at least in part by regulating the cell cycle controlling gene expression (Lv et al., 2008). The mechanisms of this antitumor activity may down-regulate the expression of Bcl-2, Survivin, Cyclin D1 and up-regulate the expression of Bax (Zhao et al., 2008).

Crocin has been proposed as the one of the antitumor ingredient of saffron. On the other hand, an interesting property of liposomes is their natural ability to target tumor site (Goyal et al., 2005; Torchilin, 2005). Leaky tumor microvasculature facilitates the accumulation of different drugs in the tumor tissues. This ability is known as the enhanced permeability and retention (EPR) effect. Nanosized liposomes can rapidly enter tumor sites from the blood vessels but these nanocarriers are kept in the bloodstream by the intact vasculature in the other tissues (Andresen et al., 2005; Farokhzad and Langer, 2009). In the present study, we decided to make nanoliposomes of crocin to improve the antitumor effect. As the cytotoxicity mechanisms of crocin are still unclear, the role of apoptosis was evaluated for both crocin and liposomal crocin.

Methods

Materials

Standard crocin was obtained from Fluka. Distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (EPC), and distearoylglycerophosphoglycerol (DSPG) were purchased from Avanti Polar Lipids . Cholesterol, chloroform, and methanol were ordered from Merck (Darmstadt, Germany). All other materials and solvents were of analytical grade.

Cell culture

The human cervical adenocarcinoma cell line (HeLa), human breast cancer cell line (MCF-7), and mouse fibroblast cell line (L929) as non-malignant cell lines were obtained from Pasteur Institute (Tehran, Iran). They were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/mL), streptomycin (100 µg/mL) at 37°C in an atmosphere of 95% humidified air and 5% carbon dioxide.

Preparation of liposomes containing crocin

Liposomal crocin was prepared from lipids and cholesterol by dehydration and rehydration (DRV) method (Malaekeh-Nikouei and Davies, 2009). In brief, liposomes were first prepared by solvent evaporation method and extruded 11 times by thermobarrel extruder (Northernlipids, Canada) through 100-nm polycarbonate filter. To 1 mL of this liposome (phospholipid concentration = 32 µmol/mL), 1 mL crocin aqueous solution was added. After freezing of this mixture by acetone and dried ice, freezed liposome was vacuum-dried in the freeze-drier for 12 h. The dried material was rehydrated by distilled water and shaked for 10 min to form liposomal suspension. Non-entrapped complex was separated by centrifugation at 14,000 rpm for 30 min and liposomes were washed twice with the same process. To prepare nanosized liposomes, liposomes were extruded repeatedly through 100-nm polycarbonate membranes at 45°C. Liposomal formulation was passed at least 11 times through the polycarbonate membrane to produce liposomes with uniform size. The composition of liposomal formulations was summarized in Table 1.

Table 1.  Composition and molar ratio of different liposomal formulations containing crocin.

DOPE	DSPG	EPC	Chol.	DSPC	Formulation type
3	—	—	3	7	I
—	3	7	7	—	II
—	3	—	7	7	III

DOPE, Dioleoylphosphatidylethanolamine; DSPG, distearoylglycerophosphoglycerol; EPC, egg phosphatidylcholine; Chol., cholesterol; DSPC, distearoylphosphatidylcholine.

Unincorporated crocin was removed three times by centrifugation at 100,000 g for 20 min. For determination of crocin concentration in the purified liposomes, aliquots of liposomes were dissolved in 20% Triton X-100 solution and the optical density was measured at 440 nm and compared with the standard curve.

Size analysis of liposomes

The mean diameter and particle size distribution of liposomes were determined using Zetasizer (NanoZS, Malvern, UK) after suitable dilution.

MTT test

The viability of cultured cells was determined by assaying the reduction of 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan. In brief, HeLa and L929 cell lines were seeded at an initial density of 5 × 10³ cells/well. MCF-7 cells were seeded 10 × 10³ cells/well, in a 96-well plate for 24 h. HeLa cells were then incubated with fresh medium containing concentrations (1, 2, 3, and 4 mM) of crocin and (0.14, 0.28, 0.42, 0.57, and 0.7 mM) of liposomal formulation I, (0.25, 0.5, 0.75, 1, 1.2 mM) of liposomal formulation II, and (1.1, 2.2, and 3.3 mM) of liposomal formulation III for 24, 48, and 72 h, respectively. The concentrations of crocin for L929 cells were 3, 5, 7, and 9 mM and for liposomal crocin were as the same of HeLa cell experiment. After incubation period, MTT was added into each well in final concentration of 0.5 mg/mL. The cells were then incubated at 37°C for 4 h. Then the insoluble formazan was collected and dissolved in dimethyl sulfoxide (DMSO, 0.5%). The absorption was measured at 570 nm (620 nm as a reference) in an ELISA reader (Start Fax-2100, UK) (Tavakkol-Afshari et al., 2008).

Propidium iodide staining

Apoptotic cells were determined by staining using the propidium iodide (PI) method described elsewhere (Zhang et al., 2004; Mousavi et al., 2009). In brief, HeLa cells were adhered overnight in a 24-well plate at the initial density of 2 × 10⁵ cells/well, and then treated with crocin and its nanoliposomes with the same concentrations for 24 h. Floating and adherent cells were then harvested and incubated overnight at 4°C in the dark with 750 µL of a hypotonic buffer (50 µg/mL PI in 0.1% sodium citrate plus 0.1% Triton X-100) before flow cytometric analysis.

Statistical analysis

Data were expressed as mean ± SD. Test was performed using one-way ANOVA followed by Tukey–Kramer post-hoc test for multiple comparisons. The P-value <0.05 was considered to be statistically significant.

Results

Table 2 shows the results of mean size and encapsulation efficiency of different liposomal formulations prepared in the present study. Mean diameters of the different liposomes containing crocin ranged between 150 and 195 nm. The highest encapsulation efficiency was achieved by liposomal formulation III.

Table 2.  Mean size and encapsulation efficiency of different liposomal formulations containing crocin (mean ± SD, n = 3).

	Formulation I	Formulation II	Formulation III
Mean diameter (nm)	194.86 ± 25.82	149.66 ± 0.92	158.83 ± 2.88
Encapsulation efficiency (%)	4.6 ± 0.8	4.8 ± 1.3	7.2 ± 2.2

Effects on cell viability

Malignant cells (HeLa and MCF-7) and L929 (as non-malignant control cells) were incubated with various concentrations of crocin (1, 2, and 4 mM) and liposomal crocin (0.5 and 1 mM) for 24, 48, and 72 h. Crocin and liposomal crocin decreased cell viability in malignant cells but not in non-malignant cells, in a concentration-dependent manner (Figures 1 and 2). This toxicity was consistent with morphologic changes including reduction in cell volume and rounding (Figure 3). Doses inducing 50% cell growth inhibition (IC₅₀) against HeLa, MCF-7, and L929 cells are presented in Table 3. Empty liposomes (without crocin) were also tested and there was no difference in cell viability in comparison with control.

Table 3.  Doses inducing 50% cell growth inhibition (IC₅₀) of crocin and its nanoliposomes against HeLa, MCF-7, and L929 cell lines.

Cell line Agent	HeLa	MCF-7	L929
Crocin	1.603	7.35	5.7
Liposomal formulation I	0.61	1.22	0.6
Liposomal formulation II	1.2	No inhibitory effect	No inhibitory effect
Liposomal formulation III	0.64	2.07	No inhibitory effect

Cells were treated with different concentrations of crocin and its nanoliposomes for 48 h. Viability was quantitated by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.

Figure 1.  Effect of crocin on cell viability of HeLa (A), MCF-7 (B), and L929 (C) cells. Cells were treated with different concentrations of crocin for 24, 48, and 72 h. Viability was quantitated by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results are mean ± SD (n = 3). The asterisks are indicator of statistical differences obtained separately at different time points compared with their controls shown in figure as *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 2.  Effect of crocin liposome 1 on cell viability of HeLa (A), MCF-7 (B), and L929 (C) cells. Cells were treated with different concentrations of crocin liposome 1 for 24, 48, and 72 h. Viability was quantitated by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results are mean ± SD (n = 3). The asterisks are indicator of statistical differences obtained separately at different time points compared with their controls shown in figure as *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3.  Morphological changes of crocin and liposomal crocin in MCF-7 and HeLa cells. HeLa and MCF-7 control cells (A and B), 48 h after treatment with 1 mM of crocin (C and D) and liposomal crocin (E and F). Control cells (A and B) remained untreated although receiving an equal volume of the solvent.

Role of apoptosis

The proportion of apoptotic cells was measured with PI staining of DNA fragmentation by flow cytometry. Crocin (1, 2, and 4 mM) and liposomal crocin (0.5 and 1 mM) induced a sub-G1 peak (one of the reliable biochemical markers of apoptosis) in flow cytometry histogram of treated cells compared with control indicating apoptotic cell death is involved in induced toxicity (Figure 4). The percent of apoptotic cells in HeLa cells is shown in Table 4.

Table 4.  The percent of apoptotic cells in HeLa cells after 48 h of crocin and its liposomal forms.

	Control	Crocin			Liposomal formulation I		Liposomal formulation II		Liposomal formulation II
Apoptosis (%)	3.8	1 mM	2 mM	4 mM	0.5 mM	1 mM	0.5 mM	1 mM	0.5 mM	1 mM
		8.3	10.7	30.8	45.6	61.5	26.7	71.5	42.1	80.6

Figure 4.  Flow cytometry histograms of apoptosis assays by propidium iodide (PI) method in HeLa cells after 24 and 48 h. HeLa cells were treated with (1, 2, and 4 mM) of crocin and (0.5 and 1 mM) of crocin nanoliposomes for 24 and 48 h. Sub-G1 peak as an indicative of apoptotic cells was induced in crocin and nanoliposome-treated but not in control cells.

Discussion

In different studies, the improvement of the pharmacokinetic properties and the pharmacological effects of liposomal drugs were reported (Chonn and Cullis, 1995; Fahr et al., 1995; Malaekeh-Nikouei et al., 2008). In the present study, the results show that the liposomal formulations containing crocin have the better cytotoxic effects compared with free crocin.

It is obvious that the carrier is an important factor for drug efficiency. Also, longer half-life of the drug in the bloodstream and better uptake by cancer cells were reported after using liposomal preparations (Andresen et al., 2005). The results of MTT test could show that crocin more effectively reached inside the cancer cells after encapsulation in liposomes. Liposomes interact with cells by different mechanisms such as adsorption, fusion, and endocytosis (Pagano and Weinstein, 1978; Kamps and Scherphof, 2003). These interactions largely related to different parameters such as the composition, size, and liposome charge (Torchilin, 2005). In this study, liposomal formulation I showed the best inhibitory effects. These fusogenic liposomes were composed of DOPE (Chonn and Cullis, 1995, 1998; Kono et al., 2000; Kunisawa et al., 2001). Using of DOPE in the liposomal formulation could change the structure of the liposome from bilayer to hexagonal, and then this conformation follows by transfer of liposome contents to cytoplasm (Pagano and Weinstein, 1978; Kamps and Scherphof, 2003). Previously, we used fusogenic liposomes for delivery of CyA to T cells. Our results showed the enhancement of immunosuppressive effects of cyclosporine A using these fusogenic liposomes (Malaekeh-Nikouei et al., 2008).

On the other hand, the size of prepared liposomes was <200 nm. As it was mentioned before, nanoparticles with a diameter up to 400 nm can accumulate in the tumor interstitium. A possible explanation for the high level of selectivity observed with these nanocarriers would be the EPR phenomena. Vascular system in tumors is irregular in shape, dilated, and contains endothelial cells with multiple defects that create “leaks.” This phenomenon makes it easier to deliver particulate delivery systems to the tumor site, which cannot normally leave the blood vessels but can extravasate into the tumor through these pores (Farokhzad and Langer, 2009).

In the present study, crocin-induced apoptosis was involved in induction of cell death. Apoptosis is a gene-regulated phenomenon, which is induced by many chemotherapeutic agents in cancer treatment (Hersey and Zhang, 2001; Debatin et al., 2002). It is characterized by distinct morphological features including chromatin condensation, cell and nuclear shrinkage, membrane blebbing, and oligonucleosomal DNA fragmentation (Green and Reed, 1998). In our study, apoptosis was determined by detection of sub-G1 peak. It has been reported that DNA fragmentation creates small fragments of DNA that can be eluted following incubation in a hypotonic phosphate–citrate buffer. When stained with a quantitative DNA-binding dye such as PI, cells that have lost DNA will take up less stain and will appear to the left of the G1 peak. The induction of apoptosis in the tumor cells is considered very useful in the management and therapy as well as in the prevention of cancer (Zhang et al., 2004). A wide variety of natural substances have been recognized to have the ability to induce apoptosis in various tumor cells (Tripathi et al., 2005). Apoptogenic effects of crocin in HeLa and MCF-7 cells have not been shown previously.

Conclusions

Altogether, the present study shows toxicity of crocin in MCF-7 and HeLa cell line with involvement of apoptosis or programmed cell death. Liposome encapsulation improved cytotoxic effects of crocin. They could be also considered as a promising chemotherapeutic agent in cancer treatment in future.

Acknowledgement

This work was supported financially by a research grant from the Vice Chancellor for Research of Mashhad University of Medical Sciences, Mashhad, Iran.

Declaration of interest

The authors declared that there is no conflict of interests in this study.