Cytotoxicity of Vinca minor

Abstract

Vinca minor L. (Apocynaceae) is a medicinal plant that has long been used to treat cerebral and memory disorders in European folk medicine. Furthermore, it contains more than 50 alkaloids, some of them having bisindole structure such as the antineoplastic alkaloids present in Catharanthus roseus (L.) G. Don (Apocynaceae). In this study, the plant’s alkaloid extract was divided into three fractions and the cytotoxic effects on cell proliferation of HT-29, Caco-2, T47D, and NIH/3T3 cell lines were examined. All alkaloid fractions showed a dose-dependent cytotoxic effect on the cell lines. IC₅₀ values confirmed that the growth and proliferation of NIH/3T3 cells were less affected in comparison to other cell lines.

Keywords::

• cytotoxicity
• fraction
• MTT
• trypan blue
• Vinca minor

Introduction

Alkaloids are an important group of diversely distributed, chemically, biologically, and commercially significant natural products. The Apocynaceae family is among the most important alkaloid-containing families, with 2664 alkaloids isolated and characterized (Cordell et al., 2001). Many plant-derived drugs to treat cancer are alkaloids. Vinca alkaloids (vinblastine and vincristine), antimitotic or antimicrotubule bisindole alkaloids derived from Catharanthus roseus (L.) G. Don (Apocynaceae), are used widely in the treatment of both childhood and adult cancers. Despite the effectiveness of these agents, drug resistance is a major clinical problem (Verrillis et al., 2003). Therefore, searching for new compounds particularly in closely related species proves invaluable.

The genus Vinca (Apocynaceae) comprises about seven species in the world. In Iran, it is represented by V. herbacea Waldst. & Kit., a native plant, with two other species being introduced or cultivated, V. minor L. and V. major L. (Mozaffarian, 2006). Vinca minor is a perennial sub-shrub, indigenous to northern Spain, through western France, eastward via central and southern Europe as far as the Caucasus; it has been naturalized in many regions. In folk medicine, it is used internally for circulatory disorders, cerebral circulatory impairment, and support for the metabolism of the brain. It is also used internally for loss of memory, hypertension, cystitis, gastritis, and enteritis, diarrhea, raised blood sugar levels, and to help weaning. It is externally used for sore throats, nosebleeds, bruising, abcesses, eczema, and to stop bleeding (Fleming, 2004).

V. minor contains eburnamine-type indole alkaloids including vincamine, which has modulatory effects on brain circulation and neuronal homeostasis as well as antihypoxic and neuroprotective potencies (Vas & Gulyas, 2005). However, few studies have been carried out on the cytotoxic activity of the plant extracts (Porska et al., 1988; Sturdikova et al., 1986).

The aim of this study was to determine the cytotoxic activity of the total alkaloid extract and the alkaloid fractions obtained from the aerial parts of the plant.

Materials and methods

Plant material

The aerial parts of V. minor were collected from Zardband Botanical Garden in Gonbad-e Kavous, Golestan Province, in June 2006. The plant was identified and authenticated by Dr. G. Amin and a voucher specimen (TEH-6654) was deposited at the Herbarium of the Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.

Tested material

Ground dried aerial parts of the plant (800 g) were extracted with MeOH–H₂O (80:20) at room temperature. The procedure was repeated until a negative test against Dragendorff’s reagent. The extracts were dried under vacuum, and the residue was treated with 2 N HCl and washed with EtOAc. The pH of the aqueous solution was adjusted to 2 by the addition of 2 N HCl and then, for each 15 mL of the solution, 0.5 g NaCl was added. It was then filtered and basified with 25% NH₃ (pH 9), and the alkaloids were extracted with CHCl₃ (10 × 300 mL). The organic fractions were combined and the solvent was evaporated under vacuum to yield the CHCl₃ alkaloid extract (11.51 g from 800 g of dried plant material). The alkaloid extract (2 g) was kept for cytotoxicity assays and the rest was initially subjected to column chromatography on silica gel, eluted with CHCl₃–MeOH (99:1, 98:2, 97:3, 96:4, 95:5, 90:10, 80:20, 70:30, 60:40, 50:50, 25:75, 10:90; 1000 mL each) to give 12 fractions. Fractions with similar thin layer chromatography (TLC) behavior were combined to yield three major fractions. Further purification was carried out with preparative TLC (20 × 20 cm, 1 mm) over silica gel eluted with CHCl₃–MeOH–25% NH₃ (17:4:0.25). Three main fractions 1 (1.83 g), 2 (0.88 g), and 3 (1.98 g) along with the total alkaloid extract (2 g) were prepared for further studies.

Cell culture

The colon carcinoma (HT-29), colorectal adenocarcinoma (Caco-2), and breast ductal carcinoma (T47D) cell lines were maintained as exponentially growing cultures in RPMI 1640 cell culture medium (PAA, Germany) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) for HT-29 cells and 15% FBS for Caco-2 and T47D cells. The Swiss mouse embryo fibroblast (NIH/3T3) cell line was kept in Dulbecco’s modified Eagle’s medium (DMEM; PAA, Germany) supplemented with 10% FBS. 100 IU/mL penicillin and 100 μg/mL streptomycin (Boehringer, Germany) were added to the media. All cell lines were cultured at 37°C in an air/carbon dioxide (95:5) atmosphere.

Determination of cell viability by MTT and Trypan blue assays

All the samples, including total alkaloid extract and three alkaloid fractions, were tested at 0.1, 1, 10, and 100 μg/mL concentrations. The samples were dissolved in dimethylsulfoxide (DMSO) and further diluted with cell culture medium. The DMSO final concentration was adjusted to 1% of the total volume of medium in all treatments, including the blank. A control without DMSO was also incubated. As positive control, 12 μM of cytarabine was used.

For MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] assay, 1 × 10⁴ cells/well were plated into 96-well plates (Nunc, Denmark) and incubated for 24 h before the addition of drugs. After 72 h of incubation for HT-29 cells, 96 h for T47D and NIH/3T3 cells, and 120 h for Caco-2 cells, 20 μL of MTT (Merck, Germany) reagent (5 mg/mL) in phosphate buffered saline (PBS) was added to each well. The plates were incubated at 37°C for 4 h. At the end of the incubation period, the medium was removed and pure DMSO (100 μL) was added to each well. The metabolized MTT product was quantified by reading the absorbance at 570 nm on a microplate reader (Anthos, Austria) (Mosmann, 1983).

For the Trypan blue assay, cells were seeded in 24-well plates (Nunc, Denmark) (3 × 10⁴ cells/well) for 24 h and were then treated with different concentrations of the samples. After the incubation period for each cell line, the medium was removed and cells were collected by Trypsinization and counted in 0.2% Trypan blue solution using a hemocytometer (Harhaji et al., 2008).

The cell viability in both MTT and trypan blue assays was calculated as a percentage of the control value (untreated cells).

IC₅₀ (The median growth inhibitory concentration) values were calculated from the IC₅₀ of dose–response curve in the SigmaPlot 10 software.

Statistical analysis

Data representative of three independent experiments with similar results are presented as mean ± SD of triplicate observations. The differences between groups of treatments were evaluated by two-way analysis of variance (ANOVA) test (SigmaStat 3.5). Values of p < 0.05 were considered to be statistically significant.

Results

Figures 1 and 2 show the viability of these cell lines when treated by Vinca minor alkaloids. Dose-dependent cytotoxicity was observed in all cell lines.

Figure 1.  The effect of total alkaloid extract and alkaloid fractions of Vinca minor on cell growth and survival. Cells (1 × 10⁴cells/well) were cultured in the absence or presence of various concentrations of alkaloid extracts, as indicated. After the indicated time points, cell viability was determined by MTT assay. (a) HT-29, (b) Caco-2, (c) T47D, (d) NIH/3T3 cells. The results are presented as mean ± SD of three triplicates.

Figure 2.  The effect of total alkaloid extract and alkaloid fractions of Vinca minor on cell growth and survival. Cells (3 × 10⁴cells/well) were cultured in the absence or presence of various concentrations of alkaloid extracts, as indicated. After the indicated time points, cell viability was determined by trypan blue assay. (a) HT-29, (b) Caco-2, (c) T47D, (d) NIH/3T3 cells. The results are presented as mean ± SD of three triplicates.

Different patterns of cytotoxicity were observed with the lower doses (0.1 and 1 μg/mL), while at higher doses most of the cells were diminished. These results suggest different sensitivities of the cell lines to the cytotoxic effect of the extracts.

Fractions 1 and 3 showed the highest activity in terms of cytotoxicity. The IC₅₀ values estimated using both methods (Table 1) were not notably different. The cytotoxic effect of the fractions on NIH/3T3 cells (as non-neoplastic cells) was weaker in comparison to other cell lines. The estimated IC₅₀ of cytarabine is reported in the Table 1.

Table 1.  Cytotoxic activity of total alkaloid extract and alkaloid fractions of Vinca minor.

Sample	Cell line^a
	HT-29		Caco-2		T47D		NIH/3T3
	T	M	T	M	T	M	T	M
Total alkaloid extract	3.49 ± 1.83	3.63 ± 1.24	0.48 ± 0.14	0.625 ± 0.12	1.10 ± 0.05	1.34 ± 0.29	3.16 ± 0.29	3.32 ± 0.16
Alkaloid fraction 1	0.47 ± 0.22	1.49 ± 0.93	0.21 ± 0.12	0.41 ± 0.14	0.29 ± 0.28	1.66 ± 0.48	2.72 ± 0.34	3.35 ± 0.23
Alkaloid fraction 2	0.74 ± 0.51	1.64 ± 0.48	0.47 ± 0.26	1.34 ± 0.84	0.78 ± 0.29	1.35 ± 0.28	2.46 ± 0.38	3.32 ± 0.05
Alkaloid fraction 3	0.45 ± 0.17	0.45 ± 0.10	0.81 ± 0.16	1.30 ± 0.42	0.67 ± 0.34	0.58 ± 0.34	2.72 ± 1.14	3.32 ± 0.08
Cytarabine	0.26 ± 0.19	0.14 ± 0.08	0.23 ± 0.15	0.16 ± 0.17	0.09 ± 0.07	0.08 ± 0.04	0.67 ± 0.34	0.74 ± 0.42

^aResults are expressed as IC₅₀ value (μg/mL), mean of three determinations. Key to cell lines employed: HT-29 and Caco-2 (colon adenocarcinoma), T47D (breast carcinoma), NIH/3T3 (Swiss embryo fibroblast). T, trypan blue method; M, MTT colorimetry.

Discussion

About 20 dimeric indole alkaloids of Catharanthus roseus have antineoplastic activity, among which vinblastine and vincristine are now available in the market.

Vinca minor is another member of the Apocynaceae family with a high content of alkaloids. Some of these alkaloids, such as vincarubine, have a bisindole structure. Although there is no study on the cytotoxic mechanism of this plant’s alkaloids, because of the structural similarity between alkaloids of Vinca minor and of Catharanthus roseus, the observed cytotoxicity could be due to the inhibition of tubulin polymerization and interference with microtubule dynamic behavior in target cells, which leads to cell cycle arrest and apoptosis (Ngan et al., 2001). The similarity of results obtained from both cytotoxic methods may indicate that the reason for cell death is neither membrane impairment nor inhibition of mitochondrial phosphorylation.

Anti-microtubule agents have additional effects on the cell; for example, these drugs may change the stereochemistry of the tubulin distal to their binding site, which then may result in downstream effects on cell function (Wang et al., 1999a).

Tubulin-binding agents also have an antivascular effect in vitro, thus affecting tumor blood flow (Hayot et al., 2002). Additional effects of tubulin-binding agents include perturbations of calmodulin (Moisoi et al., 2002), perturbation of signal transduction pathways (Wang et al., 1999b), and phosphorylation of Bcl-2, Raf-1 kinase, Bcl-xL, and p53 (Blagosklonny et al., 1997). Mitotic arrest in G1 due to induction of p21 protein independent of p53 has also been reported with these agents in some cell lines (Blajeski et al., 2002).

Vinca alkaloid-induced apoptosis may occur via a pathway independent of cell cycle arrest. These alkaloids cause significant degradation of IκBα, which in turn results in nuclear factor-kappa B (NF-κB) activation. NF-κB has been revealed to regulate the transcription of more than 150 target genes. Many of these target genes are believed to be proapoptotic genes, such as Fas/Apo-1 ligand (FasL), ICE, c-Myc, and p53 (Huang et al., 2004). However, the results obtained from this study could not reveal the mechanism of fractional cytotoxicity. Taken together with the fact that, with at least two of the fractions, dose-dependent cytotoxicity was observed may indicate a specific mechanism of cytotoxicity which needs further investigation.

The real IC₅₀ values of pure cytotoxic alkaloids in the extract may be considerably lower compared to the unpure fractions. Isolation and characterization of the alkaloid structures as well as investigation of the specific cytotoxic pathway may help to determine whether the extract is valuable for antineoplastic effects.

Declaration of interest: This research has been supported by Tehran University of Medical Sciences and Health Services grant. (Grant number: 4735-33-04-85).