Cytotoxic activity of Galantamine HBr against human cervical adenocarcinoma cell line HeLa in different concentrations (1.875 μМ ÷ 30 μМ) was evaluated. Inhibition of HeLa cell growth after treatment with Galantamine HBr and index of cell viability were determined. From the experimental results was proven that the drug exerts cytotoxic activity towards HeLa cell line, with antiproliferative activity presented as the value of IC₅₀ = 30 μM ± 0.22.

Cytotoxic activity, Galantamine HBr, Adenocarcinoma cell line HeLa

Galantamine HBr is a specific long-acting centrally active competitive (reversible) inhibitor of enzyme acetylcholinesterase (Danchev and Nikolova 2006; Ago et al. 2011), possessing positive allosteric modulatory effect on α7-subtype of nicotinic acetylcholine receptors (Arias et al. 2004; Wang et al. 2007) and antioxidant activity (Traykova et al. 2003; Tsvetkova et al. 2013a).

In accordance with these data, the aim of current study is the investigation of cytotoxic effect on human cervical adenocarcinoma cell line HeLa of Galantamine HBr, which possess acethylcholinesterase and γ-secretase inhibitory activity (Vezenkov et al. 2012) and antioxidant properties in ferric reducing/antioxidant power (FRAP) method (Tsvetkova et al. 2013b).

MTT-test of Mosmann has been widely applied for the investigation of cytotoxic activity of different compounds due to the advantages: it is rapid, versatile, quantitative, highly reproducible and sensitive. The MTT assay determines the ability of viable cells to convert a soluble yellow tetrazolium salt [3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide] (MTT) into an insoluble violet formazan crystal, that can be dissolved in an organic solvent and can be determined spectrophotometrically at λ = 570 nm. Tetrazolium salt accepts electrons from oxidized substrates or appropriate enzymes. MTT is reduced at the ubiquinone and cytochrome B and cytochrome C sites of the mitochondrial electron transport system and is the result of succinate dehydrogenase activity. The reduction takes place only when mitochondrial reductase enzymes are active and therefor conversion can be directly related to the number of viable cells. An increase in cell number results in an increase in the amount and absorbance of formazan. When the amount of formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death of cells can be deduced, through the production of a dose-response curve (Mosmann 1983).

Human epithelia cervical carcinoma cell line (HeLa) is the first type of human cancer cell lines, which has been cultured continuously. HeLa is isolated from the aggressive glandular cervical cancer of a young woman in 1951. Till now the knowledge of many processes occurring in human cells has been obtained using HeLa cells (Masters 2002).

Absorbances of positive: A(+) and negative: A(–) controls are presented on Table 1.

As a standard was used Doxorubicin. Absorbances of formazan obtained after treatment of HeLa cells with Galantamine HBr are summarized on Table 2.

The accordance between the decreased values of absorbances of formazan and concentrations of Galantamine HBr is illustrated on Fig. 2.

Figure 2. 

Effect of Galantamine HBr on the absorbances of formazan produced from HeLa cell line.

Effect of different concentrations (1.875 μМ ÷ 30 μМ) Galantamine HBr on the survival of HeLa cells is shown on Table 3.

The decreased data of index of HeLa cell viability after treatment with Galantamine HBr are presented on Fig. 3.

Figure 3. 

Effect of Galantamine HBr on HeLa cell viability.

The experimental results from the investigation of cytotoxic activity of Galantamine HBr against HeLa cells are summarized on Table 4.

The obtained data for inhibition concentration 50% (IC₅₀) are: Galantamine HBr (30 μM ± 0.22), standard Doxorubicin (3.1 μM).

The decreased growth of Hela cells treated with Galantamine HBr is illustrated on Fig. 4.

Figure 4. 

Cytotoxic activity of Galantamine HBr on HeLa cell line.

HeLa cell line was treated separately with each of the examined peptide esters in different concentrations (1.875 μМ ÷ 30 μМ) and MTT assay was evaluated. Experimental data show that the treatment of HeLa cells, resulted in reduced concentration of formazan, which indicates a growth inhibitory property of Galantamine HBr.

Inhibition of HeLa cell growth obtained after treatment with GAL is proved by the fact that in concentration 30 μM it inhibits 30.59% of cell growth with index of cell survival 69.41%. The cytotoxicity is recorded as concentration causing 50% growth inhibition (IC₅₀) of cells. It was reported that 0.1 μM Doxorubicin reduced survival of Hela cells to 40% (Sadeghi-Aliabadi et al. 2010).

Experimental results for cell growth inhibition are plotted on regression analysis using Microsoft Excel Program.

For Galantamine HBr IC₅₀ = 30 μM ± 0.22 is higher than experimental obtained data with standard Doxorubicin (IC₅₀ = 3.1 μM). This result prove that Galantamine HBr possesses lower antiproliferative activity than standard Doxorubicin.

It is reported that antitumor drug Ledakrin and other 1-nitro-9-aminoacridines produce a potent dose – dependent inhibition of DNA synthesis and replication in HeLa S3 cells by inhibition of thymidine incorporation (Woynarowski and Bartoszek 1985).

Inhibitor of phosphatidylinositol 3-kinases promotes mitotic cell death in HeLa cells (Hou et al. 2012). Anticancer activity on Hela cell lines of coumarime derivative Calanone, isolated from genus Calophyllum (IC₅₀ = 22.887 μg/ml) (Ekowati et al. 2010) and of ethanolic extract of Canthium parviflorum Lam [IC₅₀ = 43.15 μg/ml] (Purushoth et al. 2011) is proved.

It is demonstrated that the inhibitory effect on HeLa cell line of an antitumour triterpenoid: cyano enone of methyl boswellates at 1 μM is 78.5% and IC 50 = 0.27 μM. (Ravanan et al. 2011).

It is reported that DNA methyltrasferase inhibitor Zebularine (You and Park 2012) and synthetic antioxidants butylated hydroxyanisole (Moon and Park 2011) and propylgallate (Han et al. 2010) inhibit the growth of HeLa cells via caspase-dependent apoptosis, as a result of activation caspase 3 and caspase 8. The observed data for IC₅₀ are respectively: Zebularine (IC₅₀ = 130 μM) (You and Park 2012), butylated hydroxyanisole (IC₅₀ = 150 μM) (Moon and Park 2011), propylgallate (IC₅₀ = 800 μM) (Han et al. 2010). The low value of IC₅₀ correspond to the high cytotoxic activity. From the presented results it is obvious that triterpenoid is more effective in supression of cell peroliferation, followed by Zebularine, butylated hydroxyanisole and propylgallate.

(DIPP-Trp)2-Lys-OCH3 induced a dose-dependent inhibitory effect on the proliferation of HeLa cells. 100 μM (DIPP-Trp)2-Lys-OCH3 inhibits 85% of HeLa cells. The inhibitory concentration 50% (IC₅₀) is 42.23 μM (Liu et al. 2008).

In comparison with the data from our experimental results it is obvious that Galantamine HBr IC₅₀ = 30 μM is more active on HeLa cells in comparison with Zebularine, butylated hydroxyanisole and propylgallate.

Galantamine HBr treatment resulted in reduced concentration and absorbance of formazan, which indicates its growth inhibitory activity. The experimental results for inhibitory concentration IC₅₀ are 30 μM ± 0.22 and proved that Galantamine HBr exert cytotoxic activity against HeLa cell line.