Centre for pre-clinical testing of active substances laboratory for cell and molecular biology

http://cpctas.pmf.kg.ac.rs  e-mail: cpctas@kg.ac.rs

The colon cancer adenocarcinoma cell line HCT-116 was obtained from the American Tissue Culture Collection (Manassas, VA, USA). These cells were propagated and maintained in DMEM (Dulbecco’s Modified Eagle Medium), (Gibco, USA) and supplemented with 10% fetal bovine serum (PAA), antibiotics 100 IU/mL penicillin and 100 μg/mL streptomycin. Cells were growth in 75 cm² culture bottles supplied with 15 ml DMEM, and after a few passages cells were seeded in 96-well plate. Cells were cultured in a humidified atmosphere of 5% CO₂ at 37 °C.

After 24 and 72 h of treatment the cell viability was determined by MTT assay. The proliferation test is based on the color reaction of mitochondrial dehydrogenase from living cells with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma, USA). HCT-116 cells was seeded in a 96-well plate (10⁴ cells per well). After 24 h incubation cells were treated with 100 µL of each concentrations of licnenes methanol extracts (in concentration of 50-1000 µg/ml) for 24 and 72 h. Untreated cells served as a control. At the end of the tretmant period, MTT (final concentration 5 mg/ml PBS) was added to each well, wich was then incubated at 37 °C in 5% CO2 for 2 h. The colored crystals of produced formazan were dissolved in DMSO (dimethyl sulfoxide, Sigma, USA). The absorbance was measured at 550 nm. Cell proliferation was calculated as a ratio of the apsorbance of the treated group divided by the apsorbance of control group, multiplied by 100 to give percentage proliferation (Mosmann, 1983).

For analysis of cell death, we used fluorescent assays acridine orange/ethidium bromide (AO/EB) double staining. Acridine orange is taken up by both viable and nonviable cells and emits green fluorescence if intercalated into double stranded nucleic acid (DNA) or red fluorescence if bound to single stranded nucleic acid (RNA). Ethidium bromide is taken up only by nonviable cells and emits red fluorescence by intercalation into DNA. We distinguished four types of cells according to the fluorescence emission and the morphological aspect of chromatin condensation in the stained nuclei. (1) Viable cells have uniform bright green nuclei with organized structure, also have orange cytoplasm. (2) Early apoptotic cells (which still have intact membranes but have started to undergo DNA cleavage) have green nuclei, but perinuclear chromatin condensation is visible as bright green patches or fragments. (3) Late apoptotic cells have orange to red nuclei with condensed or fragmented chromatin. (4) Necrotic cells have a uniformly orange to red nuclei with organized structure (Baskic et al. 2006).

HCT-116 cells was seeded in a 6-well plate (3x10⁴ cells per well). After 24 h incubation cells were treated with 2 ml (250 μg/ml) of each licnenes methanol extracts for 24 and 72 h. Untreatd cells served as a control. Incubation was performed at 37 °C in an atmosphere of 5% CO₂ and 95% relative humidity. After 24 and 72 h of treatment, we were added 200 μl of dye mixture (100 μl of 100 mg/ml AO and 100 μl of 100 mg/ml EB in distilled water) in each well. The suspension was immediately (fast uptake) examined by fluorescence microscopy (NICON Eclipse Ti) at 400x magnification. A minimum of 300 cells was counted in every sample.

Statistical analysis

The data are expressed as the means ± standard errors (SE). Biological activity is result of three individual experiments, performed in triplicate for each dose. The magnitude of correlation between variables was done using a SPSS (Chicago, IL) statistical software package (SPSS for Windows, ver. 17, 2008). The effect of each extract were expressed by IC₅₀ (inhibitory dose which inhibit 50% growth cells) and by the magnitude of maximal effect in exposed cells. The EC₅₀ values were calculated from the dose curves by a computer program (CalcuSyn).

1. 
   Antiproliferative activity
   

Cell viability was reduced in dose-dependent manner for E. prunastri, methanol extracts. The longer time exposure induced higer cell sensitivity, except in lower concentrations (Figure 1). When compared percent of viable cells after 24 and after 72 h, cell viability was reduced in time-dependent manner for higer concentration.

The shape of dose response curves indicates a significant inhibition of cell growth in dose-dependent and time-dependent manner for H. physodes, F. caperata, C. foliacea (Figure 2, Figure 4 and Figure 5). Our results show better activity in higer concentration. The longer time exposure induced higer cell sensitivity (Figure 2, Figure 4 and Figure 5).

The shape of dose response curves indicates a not significant inhibition of cell growth in dose-dependent manner for P. sulcata (Figure 3). Maximal inhibition after 24 and 72-hours exposure was observed for concentration of 1000 μg/ml for P. sulcata extracts.

The longer time exposure induced lower cell sensitivity (Figure 3). When compared percent of viable cells after 24 and after 72 h, cell viability was not reduced in time-dependent manner for all concentration.

According to the American National Cancer Institute (NCI), the criteria of cytotoxic activity for the active substances extracted from Evernia prunastri, Hypogimnia physodes, Parmelia sulcata, Flavoparmelia caperata, Cladonia foliacea were completely unable to inhibit HCT-116 cell growth at the used concentrations (IC₅₀ >30 μg/ml) (Table 1) and this extracts demonstrated an intermediate activity after 24 and 72 h.

The data resulting from three different experiments are summarized in Table 1.
2. The results obtained with AO/EB double staining

The ability of the lichens extracts to induce apoptosis was initially screened by using acridine orange/ ethidium bromide staining. Control cells showed bright green nucleus with uniform intensity and had not taken up ethidium bromide, where the apoptotic cells appeared orange in color (Figure 6 and Figure 7). The all lichens extract treated cells showed obvious nuclear condensation after 24 h treatment. Florescence microscopic images clearly showed nuclear disintegration of cells which were treated by methanolic extracts of all lichenes species compared with that of the untreated control cells when stained with acridine orange and ethidium bromide. Compared with spontaneus apoptosis observed in control cells (early apoptotic 3.2%, 0% late apoptotic and 0% necrotic cells) HCT-116 was treated by 250 µg/ml methanolic extract of all lichenes showed increased percentages of early apoptotic for 24 h (Figure 6). The methanolic extract of H. physodes wich exhibited the highest antiproliferative potential with IC₅₀ of 253.72 μg/ml (Table 1) showed increased percentages of early apoptotic (42.22%), increased percentage of late apoptotic (11.11%) and icreased necrose (17.78%) cells after 24 h (Figure 6). Methanol extract C. foliacea showed maximal effect on icreased early apoptotic (49.66%). Compared with the spontaneous apoptosis observed in control cells (early apoptotic 28.8% after 72 h), HCT 116 treated with 250 μg/ml dose of different lichens extract showed slightly increased percentages of late apoptotic (Figure 7). The all lichens extract treated cells showed obvious nuclear condensation after 72 h treatment. Our results showed increase of late apoptosis (53.15%) and significant increase of necrosis (32.62%) cells treated with methanol extract of H. physodes after 72 h (IC₅₀=102.405μg/ml). Methanolic extract of C. foliacea showed increased percentages of late apoptotic (48.4%), cells after 72h (Figure 7).

Based on the above cytomorphological changes and cell death the effect of methanol extract in these cells were indicative of early apoptosis after 24 h and late apoptosis after 72 h.

To further determine the activity and mechanism of action of the methanol extract, we would like to isolate and identify the active principle and investigate the effects in more detail.

Baskić D, Popović S, Ristić P, Arsenijević NN. Analysis of cycloheximide-induced apoptosis in human leukocytes: Fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide. Cell Biol Int 2006; 30(11): 924-932.

Itharat A, Houghton PJ, Eno-Amooquaye E, Burke PJ, Sampson JH, Raman A. In vitro cytotoxic activity of Thai medicinal plants used traditionally to treat cancer. J Ethnopharmacol. 2004; 90: 33–38.

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