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Proapoptotic and Antimigratory Effects of Pseudevernia furfuracea and Platismatia glauca on Colon Cancer Cell Lines

SUMMARY The aim of this study is to investigate cytotoxic, proapoptotic, antimigratory and pro-antioxidant effects of methanol, acetone and ethyl acetate extracts of lichens Pseudevernia furfuracea and Platismatia glauca on colorectal cancer (HCT-116 and SW-480) cell lines. We compared the cytotoxic effects on colorectal cancer cells with the effects obtained from normal human fibroblast (MRC-5) cell line. Tetrazolium (MTT) test evaluated the cytotoxic effects, Transwell assay evaluated cell migration, acridine orange/ethidium bromide (AO/EB) fluorescent method followed the apoptosis, while prooxidant/antioxidant effects were determined spectrophotometrically through concentration of redox parameters. The tested extracts showed considerable cytotoxic effect on cancer cells with no observable cytotoxic effect on normal cells. Ethyl acetate and acetone extract of P. furfuracea induced the highest cytotoxicity (IC50=(21.2±1.3) µg/mL on HCT-116, and IC50=(51.3±0.8) µg/mL on SW-480 cells, respectively, after 72 h), with noteworthy apoptotic and prooxidant effects, and antimigratory potential of methanol extract. P. glauca extracts induced cytotoxic effects on HCT-116 cells after 72 h (IC50<40 μg/mL), while only methanol and acetone extracts had cytotoxic effects on SW-480 cells after 24 h, with proapoptotic/necrotic activity, as a consequence of induced oxidative stress. In conclusion, lichen extracts changed to a great extent cell viability and migratory potential of colorectal cancer cell lines. HCT-116 cells were more sensitive to treatments, P. furfuracea had better proapoptotic and antimigratory effects, and both investigated lichen species might be a source of substances with anticancer activity.


INTRODUCTION
Colorectal cancer (CRC) is among major causes of mortality in the world, and the second cause of cancer-related death (1). Carcinogenesis is a multistage process with progressive changes at the cellular, genetic and epigenetic level. The main characteristics of cancer cells are proliferation, inactivated tumour suppressor genes, suppressed and inactivated genes and pathways responsible for cell death and capacity to migrate to other organs (2). Generally, treatment options for CRC patients are surgery, chemotherapy and radiation. Novel anticancer therapeutic strategies find basis in the activation of programmed cell death (apoptosis) and the suppression of cell migration (2). The activation of apoptosis is a key molecular mechanism responsible for anticancer activities of most of the currently studied potential anticancer agents (2). Oxidative stress and reactive oxygen species (ROS) as signal molecules have a major role in many physiological and pathological processes in cells (3). Overproduction of ROS induces the cytotoxic activity and apoptosis induction by various anticancer agents (e.g. cisplatin, camptothecin, inostamycin, doxorubicin and mitomycin C) (4), and some active compounds isolated from natural sources (5). Also, the reduction of cell migration, as a critical process in cancer development, depends on the production of ROS and reactive nitrogen species (RNS) in the treatments with anticancer drugs (6).
Natural sources (plants, mushrooms and lichens) contain different active substances with antitumour activity. Great number of drugs isolated from natural sources are approved for use in traditional medicine (7). Lichens have been widely used in the folk medicine as laxatives and antiemetic, which indicates their application in treatments of gastrointestinal disorders (8). Lichens synthesize a plenty of secondary metabolites with diverse biological activities (9). Also, many studies have shown that extracts of lichen species obtained from different areas in Serbia had antioxidant, antimicrobial and antiproliferative potential (10-12). Pseudevernia furfuracea (L.) Zopf. demonstrated a notable cytotoxic effect (in acetone extract) on the FemX (human melanoma) and LS174 (human colon carcinoma) cell lines (11). In addition, Platismatia glauca (L.) W.L. Culb. & C.F. Culb. had considerable cytotoxic effects (in n-hexane extracts) on L1210 (murine leukemia), 3LL (murine Lewis lung carcinoma) and U251 (human glioblastoma) (13). The literature data confirm that some active substances, such as acids isolated from lichens, can induce apoptosis in murine leukemia L1210 cell (13).
Recently, Mitrović et al. (14) have shown that extracts of P. glauca and P. furfuracea had antioxidant, antimicrobial and antibiofilm effects, and also gave the chemical profile of these extracts. The goal of this study is to evaluate cytotoxic, proapoptotic, antimigratory and pro-antioxidant impact of the methanol, acetone end ethyl acetate extracts of P. furfuracea and P. glauca lichen species from Serbia on two colorectal cancer cell lines (HCT-116 and SW-480). For estimation of antitumour effects, we also evaluated cytotoxicity of lichen extracts on normal human fibroblast MRC-5 cell line.

Cell culture
The human colon cancer HCT-116, SW-480 and normal human fibroblast MRC-5 cell lines were obtained from the American Tissue Culture Collection, Manassas, VA, USA. The cells were maintained and seeded in a 96-well plate as described by Šeklić et al. (16). MRC-5 cell line was seeded only for tetrazolium (MTT) assay. Untreated cells served as negative control. The results for MTT, acridine orange/ethidium bromide (AO/EB) staining, nitroblue tetrazolium (NBT), Griess and glutathione (GSH) tests were obtained after 24 and 72 h. For Transwell migration assay the results were measured 24 h after incubation.

Cytotoxicity assay
Cell viability was determined by MTT assay as described by Šeklić et al. (16). The concentrations of the three used lichen extracts (methanol, acetone and ethyl acetate) were 1, 10, 50, 100, 250 and 500 µg/mL. The results were presented as percentage of viability (ratio between the absorbance of treated and control cells). IC 50 values (the extract dose that inhibited cell growth by 50 %) served as the measure of cytotoxicity and were calculated by CalcuSyn v. 2.1 (17).

Fluorescence microscopic analysis of cell death
For the analysis of the stages of cell death in control and treated cells, we used AO/EB and double staining fluorescent microscopic assays according to the procedure described by Mitrović et al. (12). For this method, HCT-116 and SW-480 cells were treated with 100 µL of each extract at two selected concentrations (10 and 100 µg/mL). The cells were observed under the fluorescence microscope Eclipse Ti (Nikon Instruments Inc., Melville, NY, USA) at 400× magnification using Nikon NIS-Elements Advanced Research software v. 3.2 (18).

Transwell migration assay
Transwell migration assay (19) measured the effects of lichen extracts on the cell migration. Šeklić et al. (16) described a detailed protocol. The HCT-116 and SW-480 cells were treated with two selected concentrations (10 and 50 µg/mL) of methanol lichen extracts, and the results were presented as index of migration/absorbance (A) (19), and (index of migration/A)/N(viable cells), respectively.

Determination of redox status parameters
Monitoring the concentrations of superoxide anion radical, nitrites and reduced glutathione (GSH) following detailed protocols described by Šeklić et al. (16) helped determine the prooxidant/antioxidant status. These parameters were determined in the four selected concentrations of each extract (1, 10, 50 and 100 µg/mL).
The colour reaction in MTT, Transwell, NBT, nitrites and GSH tests was measured spectrophotometrically on RT-2100C ELISA microplate reader (Hamburg, Germany). The results for NBT, nitrites and GSH tests were expressed in µM per number of viable cells on the basis of data given in MTT test according to Živanović et al. (20).

Statistics
All data are presented as the mean value±standard error (S.E.) of three independent experiments, in triplicate for each dose. Student's t-test and one-way ANOVA for multiple comparisons evaluated statistical significance using SPSS for Windows, v. 17 (21).

RESULTS AND DISCUSSION
Cytotoxic activity Fig. 1 shows the effects of treatments with methanol, acetone and ethyl acetate extracts of Pseudevernia furfuracea and Platismatia glauca on colorectal cell viability, while Table 1 shows cytotoxic activities of the tested extracts, expressed via IC 50 values. All treatments considerably decreased cell viability in dose-and time-dependent manner (except the P. glauca extracts in lower dose on SW-480 cells) of tested cancer cell lines ( Fig. 1) and had moderate effect on normal MRC-5 cells ( Table 1).
The most significant cytotoxic effect was observable of ethyl acetate extract of P. furfuracea on HCT-116 cells (IC 50 =(21.2±1.3 IC 50 μg/mL)) after 72 h. Acetone extract of P. furfuracea after 72 h had the highest cytotoxic effect on SW-480 cells. All extracts of P. glauca demonstrated significant cytotoxic effect (IC 50 <40 μg/ mL) on HCT-116 cell line after 72 h, while methanol and acetone extracts of P. glauca had significant cytotoxic effects on SW-480 cells after 24 h. The literature data defined criteria for cytotoxicity of the extracts as IC 50 <30 μg/mL (22) and according to this criterion, ethyl acetate extract of P. furfuracea expressed significant cytotoxicity on HCT-116 cell line. As our results showed, all tested samples demonstrated cytotoxic effect on both cell lines, while HCT-116 cells were more sensitive to treatments.  Concerning the results obtained with P. glauca, the present study revealed significant cytotoxicity of all tested extracts on HCT-116 cells after 72 h, while the highest effect of methanol and acetone extracts was on SW-480 cells after 24 h. Based on the obtained results, we can conclude that the P. glauca extract induces a significantly acute cytotoxic effect. The literature data show that extracts of lichen may have significant cytotoxic effects on colon cancer cells after 24 h (23). The investigated treatment with P. glauca in low concentrations activated proliferation in survivor cells and the effects of proliferation were measured after 72 h (higher IC 50 values after 72 h).
In our earlier study, chemical profiling of P. glauca extract revealed dimethyl caperate, atraric acid, chloratranol and atranol as major components (14). We must be aware of the presence of other minor components (5,7-dihydroxy-6-methylphtalide, isoadiantone, olivetol, methyl and ethyl haematommate) and their hidden contribution to the detected cytotoxic activity.
However, normal human fibroblast MRC-5 cell line is modestly sensitive to treatments (IC 50 >200 μg/mL) ( Table 1), thus indicating cell-specific activities of tested lichen extracts. According to the data presented in our study, P. furfuracea and P. glauca extracts might be considered a possible source of anticancer agents.

Proapoptotic effects
Our results show that the tested extracts induced cell death mainly by apoptosis. The treated cells showed morphological changes typical for apoptosis (reduction of cell size, blabbing effects with condensation and fragmentation of nuclear material, and formation of apoptotic bodies) (Fig. 2). Table 2 shows the percentages of viable, early apoptotic, late apoptotic and necrotic cells in HCT-116 and SW-480 cell lines, respectively. The number of viable cells detected with MTT assay (Fig. 1) correlates with the number of viable and early apoptotic cells observed by microscopic AO/EB double staining, as a more sensitive assay. The literature data (23) confirm that cells in the early apoptosis still have the capacity to metabolize the tetrazolium salt (the main component of the MTT test), and according to the MTT test, they are detected as living. It is also known that cellular adhesion is not impaired in the early apoptosis, so the cells at this stage are still attached to the plate, i.e. they are detected as alive (23). The changes of molecules responsible for  According to the data presented in Table 2, apoptotic effects depend on the concentration of treatments and time of exposure. Stronger effects were observed after longer exposure time (72 h), except for P. glauca extract tested on SW-480 cells, which showed better results after 24 h ( Table 2). The ethyl acetate extract of P. furfuracea, with the strongest cytotoxic effects at HCT-116 cells after 72 h, showed a significant apoptotic effect at both tested doses (10 µg/mL caused 20.12 % of late apoptotic cells, while 100 µg/mL induced 75.39 % of late apoptotic cells and 5.62 % of necrotic cells; Table 2). A significant percentage of viable cells detected by MTT were in the early apoptosis stage (41.37 and 10.94 % in the presence of 10 and 100 µg/mL ethyl acetate extract of P. furfuracea, respectively), indicating that this lichen extract possessed very strong proapoptotic potential against HCT-116 cells. The extracts of P. glauca induced significant, dose-and time-dependent proapoptotic effects on HCT-116 cells. However, the acetone and ethyl acetate extracts of P. glauca induced modest necrotic effects on the same cells. Regarding the induced apoptosis on HCT-116 cells, the extracts of P. furfuracea could be ranged as follows: ethyl acetate>acetone>methanol, and of P. glauca: methanol>ethyl acetate>acetone ( Table 2).
The results presented in Table 2 show that methanol and acetone extracts of P. furfuracea had the highest proapoptotic effects on SW-480 cells after 72 h, which was in accordance with the results of the cytotoxicity (IC 50 values in Table 1). The methanol and acetone extracts of P. glauca induced higher apoptotic effects on SW-480 cells after 24 h of exposure ( Table 2). Regarding the induced apoptosis in SW-480 cells, P. furfuracea extracts could be ranged as follows: acetone>methanol>ethyl acetate, and of P. glauca: methanol>acetone>ethyl acetate (Table 2). All P. furfuracea extracts had modest necrotic effects on SW-480 cells (3.49-7.49 %), but only in higher dose (100 µg/ mL) after 72 h. However, P. glauca extracts demonstrated the strongest necrotic effects on the same cells, in both tested doses and treatment periods ( Table 2). The methanol and acetone extracts of P. glauca had the highest necrotic potential after 24 h, and together with induced apoptosis, these data clearly coincided with the cytotoxic activity (IC 50 values in Table 1) and may be a consequence of oxidative stress (24). Generally, the cytotoxic effects of P. furfuracea extracts were the consequence of induced apoptosis in HCT-116 and SW-480 cells, while P. glauca extracts induced apoptosis and necrosis in both cell lines, especially 72 h after treatments. According to the presented data, lichen extracts with the highest cytotoxic effects had the strongest proapoptotic effects on the tested cancer cell lines.
A few studies (12,13,23) demonstrate the induction of apoptosis in cancer cells by lichen metabolites. Apoptosis, as a strongly regulated process of programed cell death, can be induced via two main pathways, the extrinsic (death receptor pathway) and the intrinsic (mitochondrial pathway) (25). The literature data show that the treatment with lichen extracts induces the genomic DNA fragmentation with significant elevation of caspase 3 activities in cancer cells (26). All tested extracts, especially P. furfuracea extracts, induce nuclear condensation and cell damage in apoptotic process, indicating that the tested extracts have a potential to eliminate tumour cells by apoptosis. According to the obtained results, we can conclude that HCT-116 cells are more sensitive to proapoptotic effects of lichen extracts.

Migratory potential
Cell migration is a key problem during the development and progression of cancer. Thus, we evaluated antimigratory potential of methanol extracts of P. furfuracea and P. glauca on HCT-116 and SW-480 cells. The methanol extract was selected because it had the weakest cytotoxicity on the tested cell lines after 24 h (except the extracts of P. glauca on SW-480 cells). The 10 and 50 µg/mL methanol extracts of tested lichens considerably decreased migration of HCT-116 and SW-480 cells (Fig. 3). After the evaluation of absorbance related to the number of viable cells, all methanol extracts of P. furfuracea and P. glauca showed noteworthy antimigratory effects on HCT-116 cells. Further, the tested extracts showed antimigratory effects on the SW-480 cells, except the P. glauca extract at the lower concentration (10 µg/mL), thus demonstrating promigratory effect. As the literature data confirmed, a significant change in the prooxidant/antioxidant balance could have an impact on the stimulation of cellular mobility (27,28). On the other hand, P. glauca extract induced multiple increase in GSH in SW-480 cells, indicating an antioxidant effect. A high level of GSH in cancerous cells allowed them to survive oxidative stress by favouring the survival of cells with high invasive and metastatic potential (28,29).
P. furfuracea extract (50 µg/mL) revealed the best antimigratory effects in both cell lines (Fig. 3). In the literature, there are a few studies that focus on the effects of whole polyphenol extracts on migration and metastatic potential of cancer in vitro (30,31). To the best of our knowledge, there are no data about the effects of the tested lichen species extracts on cell migratory potential, and our study is the first to show the antimigratory potential of P. furfuracea and P. glauca on colorectal cancer HCT-116 and SW-480 cell lines. Our results suggest stronger antimigratory capacity of P. furfuracea than P. glauca probably according to their chemical profile. The comparison of the obtained results revealed that methanol extract of P. furfuracea was richer in phenols than the same extract of P. glauca (14), thus suggesting that the phenols participated in antimigratory effects on the tested cancer cells. The literature data confirmed that polyphenolic compounds could suppress cell migration and reverse metastatic cells (31,32 The results presented in this work indicate that HCT-116 cells are more sensitive to antimigratory effects of the investigated lichen species than SW-480 cells, which agrees with our earlier findings on migratory potential of these cell lines (33).

Redox status
As a very potent and significant member of reactive oxygen species (ROS), the superoxide anion radical indicates the occurrence of oxidative stress in supraphysiologically high concentration. Fig. 4. shows the effects of methanol, acetone and ethyl acetate extracts of P. furfuracea and P. glauca on O 2 •level in HCT-116 and SW-480 cells, expressed per number of cells. Generally, all treatments were prooxidative, with increased O 2 •level, mainly in dose-and time-dependent way. Higher concentrations of extracts (50 and 100 µg/mL) considerably increased O 2 •levels in both cell lines, and the highest prooxidative effect was observed in P. furfuracea (Fig. 4). High concentrations of nitrites (as an indicator of nitric oxide) could be a parameter of nitrosative stress in cells. We investigated the nitrite level and showed the obtained data in Fig. 5. Generally, all tested lichen extracts showed doseand time-dependent increase of nitrite level in HCT-116 cells. In lower doses, this increase of nitrites was not as prominent as in higher doses. P. furfuracea extracts induced greater nitrite increase than P. glauca extracts. On the other hand, we also observed nitrite increase in SW-480 cells, but in a slightly different way. Lower concentrations of P. furfuracea induced very slow nitrite elevation with the increase of applied extract doses, and only the highest dose induced extreme nitrite rise, especially after 72 h at treatment. On the other hand, after 24 h of treatment, P. glauca induced greater increase at lowest concentrations, thus reaching the plateau of effect with almost no change in the increase of extract doses. After 72 h, the increase of nitrites was dose-dependent. P. glauca induced greater nitrite levels after 24 h than after 72 h. We conclude that P. furfuracea had greater impact on the tested cell lines than the lichen species.
The reduced glutathione (GSH) content is considered a very important parameter of cell antioxidative capacity. In our experiments, all treatments predominantly induced decrease of GSH at low concentrations and increase in higher concentrations, depending on a lichen species, type of extract, concentration and treatment period (Fig. 6). P. furfuracea induced greater increase of GSH level in the investigated cells, especially in SW-480 after 72 h of treatment. P. glauca induced greater increase in HCT-116 cells, while the investigated extracts induced notable GSH increase in SW-480 cells, in low doses and after 24 h of treatment.
Considering the given parameters of redox status in relation to the number of viable cells, we concluded that the treated cancer cells were in oxidative stress. All the measured parameters (O 2 •-, NO 2 and GSH) considerably increased at higher concentrations, mainly 72 h after treatments. P. furfuracea induced higher oxidative stress in both tested cell lines than P. glauca extracts.
It is known that lichen extracts and purified components could change oxidative characteristics of cancer cells and that the ROS overproduction has important role in the cytotoxicity, the initiation and execution of apoptosis (4,26). According to our results, the investigated cells were under great oxidative/nitrosative stress, which induced enhanced cytotoxicity through apoptosis. These effects were the most prominent with P. furfuracea extracts. De novo generated nitrites spontaneously combine with O 2 •and generate peroxynitrites, which are considered very reactive and toxic reactive nitrogen species (RNS) with notable cytotoxic and proapoptotic effects (33). Our results showed that prooxidative capacity (increased O 2 •-) of P. furfuracea and P. glauca methanol extracts directly correlated with their antimigratory effects on the treated colon cancer cell lines. The literature data (6) confirmed that ROS, in the first instance O 2 •and H 2 O 2 , could reduce migratory potential of cancer cells.
Glutathione as a parameter of antioxidative protection of cells is the most abundant redox pool of cells, and depletion of reduced glutathione content could be the consequence of antioxidant response of the tested cells to the increased oxidative stress (34). Our results show that GSH content increased in HCT-116 and more evidently in SW-480 cells. These findings suggest that the tested colorectal cell lines had different response to the same treatment. As a consequence of