In vitro evaluation of cytotoxic, anti-proliferative, anti-oxidant, apoptotic, and anti-microbial activities of Cladonia pocillum

M Ersoz; Z. M. Coskun; B. Acikgoz; I. Karalti; G. Cobanoglu; C. Cesal

doi:10.14715/cmb/2017.63.7.12

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Vol. 63 No. 7: Issue 7

Issue Published : August 22, 2017

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In vitro evaluation of cytotoxic, anti-proliferative, anti-oxidant, apoptotic, and anti-microbial activities of Cladonia pocillum

https://doi.org/10.14715/cmb/2017.63.7.12

M Ersoz

Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Istanbul Bilim University, Istanbul, Turkey

Z. M. Coskun

Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Istanbul Bilim University, Istanbul, Turkey

B. Acikgoz

Department of Biology, Science and Art Faculty, Marmara University, Istanbul, Turkey

I. Karalti

Clinic Microbiology Laboratory, Medical Faculty, Yeditepe University, Istanbul, Turkey

G. Cobanoglu

Department of Biology, Science and Art Faculty, Marmara University, Istanbul, Turkey

C. Cesal

Department of Biology, Science and Art Faculty, Marmara University, Istanbul, Turkey

Corresponding Author(s) : M Ersoz

mersoz1@yahoo.com

Cellular and Molecular Biology, Vol. 63 No. 7: Issue 7
Article Published : August 15, 2017

Abstract

The aim of this study was to investigate the anti-proliferative, apoptotic, cytotoxic, and anti-oxidant effects of extracts from the lichen Cladonia pocillumon human breast cancer cells (MCF-7), and to characterize the anti-microbial features. MCF-7 cells were treated with methanolic C. pocillum extract for 24h. The cytotoxicity of the extract was tested with MTT. Moreover, its anti-proliferative effects were examined with immunocytochemical method. Apoptosis and biochemical parameters were detected in MCF-7. The methanol and chloroform extracts of the lichen were tested for anti-microbial activity against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans using the disc diffusion method and calculation of minimal inhibitory concentrations. Although BrdU incorporation was not observed in MCF-7 cells treated with methanol extract at a concentration above 0.2 mg/mL, a significant decrease was observed int he percentage of PCNA immunoreactive cells in groups treated with 0.2, 0.4, 06, and 0.8 mg/mL methanol extracts of C.pocillum (49±6.3, 44±5.2, 23±2.5, 0, respectively) compared to that of control (85±4.5). The percentage of apoptotic cells significantly increased in groups treated with 0.2, 0.4, 0.6, and 0.8 mg/mL extracts of the C.pocillum (54±3.5, 76±2.6, 77±1.8, 82±4.2, respectively) compared with that of control group (3.9±1.5).The half-maximal inhibitory concentration of the methanol extract against MCF-7 cells was 0.802 mg/mL .Although the chloroform extract showed more effective anti-microbial activity overall, the methanol extract showed higher anti-fungal activity. Collectively, the results of our study indicate that C.pocillum extracts have strong anti-microbial and apoptotic effects. This lichen therefore shows potential for development as a natural anti-microbial, anti-oxidant, and apoptotic agent.

Keywords

Cell proliferation Lichen Microbial activity MCF-7 TUNEL.

Ersoz, M., Coskun, Z. M., Acikgoz, B., Karalti, I., Cobanoglu, G., & Cesal, C. (2017). In vitro evaluation of cytotoxic, anti-proliferative, anti-oxidant, apoptotic, and anti-microbial activities of Cladonia pocillum. Cellular and Molecular Biology, 63(7), 69–75. https://doi.org/10.14715/cmb/2017.63.7.12

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References

Dayan FE, Romagni JG. Lichens as a potential source of pesticides. Pesticide Outlook 2001; 12:229-32.
Suleyman H, Yildirim D, Aslan A, Gocer F, Gepdiremen A, Guvenalp Z. An investigation of the anti-inflammatory effects of an extract from Cladonia rangiformis. HOFFM Biol Pharm Bull 2002; 25:10-3.
Karunaratne V, Bombuwela K, Kathirgamanathar S, Thadhani VM. Lichens: a chemically important biota. J Natn Sci Foundantion Sri Lanka 2005; 33:169-86.
Oran S, Sahin S, Sahinturk P, Oztürk S, Demir C. Antioxidant and Antimicrobial Potential, and HPLC Analysis of Stictic and Usnic Acids of Three Usnea Species from Uludag Mountain (Bursa, Turkey). Iran J Pharm Res 2016; 15:527-35.
Siegel R, Naishadham D, Jemal A. Cancer statistics. CA Cancer Journal for Clinicians 2013; 63:11-30.
Sosa V, Moliné T, Somoza R, Paciucci R, Kondoh H, LLeonart ME. Oxidative stress and cancer: an overview. Ageing Res Rev 2013; 12(1):376-90.
Martinez-Useros J, Li W, Cabeza-Morales M, Garcia-Foncillas J. Oxidative Stress: A New Target for Pancreatic Cancer Prognosis and Treatment. J Clin Med 2017; 6(3):E29.
Hecht F, Pessoa CF, Gentile LB, Rosenthal D, Carvalho DP, Fortunato RS. The role of oxidative stress on breast cancer development and therapy. Tumour Biol 2016; 37(4):4281-91.
Ren MR, Hur JS, Kim JY, Park KW, Park SC, Seong CN, et al. Anti-proliferative effects of Lethariella zahlbruckneri extracts in human HT-29 human colon cancer cells. Food Chem Toxicol 2009; 47:2157-62.
Manojlović N, Ranković B, Kosanić M, Vasiljević P, Stanojković T. Chemical composition of three Parmelia lichens and antioxidant, antimicrobial and cytotoxic activities of some their major metabolites. Phytomedicine 2012; 19:1166-72.
Brandí£o LF, Alcantara GB, Matos Mde F, Bogo D, Freitas Ddos S, Oyama NM, et al. Cytotoxic evaluation of phenolic compounds from lichens against melanoma cells. Chem Pharm Bull 2013; 61:176-83.
Su ZQ, Liu YH, Guo HZ, Sun CY, Xie JH, Li YC, et al. Effect-enhancing and toxicity-reducing activity of usnic acid in ascitic tumor-bearing mice treated with bleomycin. Int Immunopharmacol. 2017; 46:146-55.
Bézivin C, Tomasi S, Lohézic-Le Dévéhat F, Boustie J. Cytotoxic activity of some lichen extracts on murine and human cancer cell lines. Phytomedicine 2003; 10:499-503.
Baćkorová M, Jendželovskí½ R, Kello M, Baćkor M, Mikeš J, Fedoroćko P. Lichen secondary metabolites are responsible for induction of apoptosis in HT-29 and A2780 human cancer cell lines. Toxicol In Vitro 2012; 26:462-8.
Subedi A, Amatya MP, Shrestha TM, Mishra SK, Pokhrel BM. Antioxidant and antibacterial activity of methanolic extract of Machilus odoratıssıma. KUSET 2012; 8:73-80.
Smith CW, Aptroot A, Coppins BJ, Fletcher A, Gilbert OL, James PW, et al. The lichens of Great Britain and Ireland. The British Lichen Society, London, 2009, pp.309-38.
Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1963; 61:882-8.
Ledwozyw A, Michalak J, StepieÅ„ A, KadzioÅ‚ka A. The relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis. Clin Chim Acta 1986; 155(3):275-83.
Aebi H. Catalase in vitro. Methods Enzymol 1984; 105:121–6.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193(1):265-75.
Bauer AW, Kirby WMN, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized simple disc method. Am J Clin Pathol 1966; 45:493.
Fridlender M, Kapulnik Y, Koltai H. Plant derived substances with anti-cancer activity: from folklore to practice. Front Plant Sci. 2015; 6:799
Acikgoz B, Karalti I, Ersoz M, Coskun ZM, Cobanoglu G, Sesal C. Screening of antimicrobial activity and cytotoxic effects of two Cladonia species. Z Naturforsch C 2013; 68:191-7.
Mitrović T, Stamenković S, Cvetković V, Tošić S, Stanković M, Radojević I, et al. Antioxidant, antimicrobial and antiproliferative activities of five lichen species. Int J Mol Sci 2011; 12:5428-48.
Ranković BR, Kosanić MM, Stanojković TP. Antioxidant, antimicrobial and anticancer activity of the lichens Cladonia furcata, Lecanora atra and Lecanora muralis. BMC Complement Altern Med 2011; 11:97-104.
Singh N, Nambiar D, Kale RK, Singh RP. Usnic acid inhibits growth and induces cell cycle arrest and apoptosis in human lung carcinoma A549 cells. Nutr Cancer 2013; 65:36-43.
Kosanić MM, Ranković BR, Stanojković TP. Antioxidant, antimicrobial and anticancer activities of three Parmelia species. J Sci Food Agric 2012; 92:1909-16.
Kosanić M, Ranković B, Stanojković T. Antioxidant, antimicrobial, and anticancer activity of 3 Umbilicaria species. J Food Sci 2012; 77:T20-5.
Basile A, Rigano D, Loppi S, Di Santi A, Nebbioso A, Sorba Sergio, et al. Antiproliferative, antibacterial and antifungal activity of the lichen Xanthoria parietina and its secondary metabolite parietin. Int J Mol Sci 2015; 16:7861-75.
Levkoff LH, Marshall GP 2nd, Ross HH, Caldeira M, Reynolds BA, Cakiroglu M, et al. Bromodeoxyuridine inhibits cancer cell proliferation in vitro and in vivo. Neoplasia. 2008; 10(8):804-16.
Stoimenov I, Helleday T. PCNA on the crossroad of cancer. Biochem Soc Trans. 2009; 37(Pt 3):605-13.
Romagni JG, Dayan FE. Structural diversity of lichen metabolites and their potential use. In: Advances in microbial toxin research and its biotechnological exploitation. Upadhyay RK. (ed), Kluwer Academic/Plenum Publishers, New York, 2002, pp.151-70.
Mayer M, O'Neill MA, Murray KE, Santos-Magalhaes NS, Carneiro-Leao AM, Thompson AM, et al. Usnic acid: a non-genotoxic compound with anticancer properties. Anti Canc Drugs 2005; 16:805-9.
Brisdelli F, Perilli M, Sellitri D, Piovano M, Garbarino JA, Nicoletti M, et al. Cytotoxic activity and antioxidant capacity of purified lichen metabolites: an in vitro study. Phytother Res. 2013; 27(3):431-7.
Yilmaz M, Ozdemir Turk A, Tay T, Kivanc M. The antimicrobial activity of extracts of the lichen Cladonia foliacea and its (–)-usnic acid, atranorin, and fumarprotocetraric acid constituents. Z Naturforsch C 2004; 59:249-54.
Ingolfsdottir K. Molecules of interest – usnic acid. Phytochemistry 2002; 61:729-36.
Cobanoglu G, Sesal C, Gokmen B, Cakar S. Evaluation of the antimicrobial properties of some lichens. South West J Hortic Biol Environ 2010; 1:153-8.
Ranković B, Mišic M, Sukdolak S. Antimicrobial activity of extracts of the lichens Cladonia furcata, Parmelia caperata, Parmelia pertusa, Hypogymnia physodes and Umbilicaria polyphylla. Biologia 2009; 64:53-8.

References

Dayan FE, Romagni JG. Lichens as a potential source of pesticides. Pesticide Outlook 2001; 12:229-32.

Suleyman H, Yildirim D, Aslan A, Gocer F, Gepdiremen A, Guvenalp Z. An investigation of the anti-inflammatory effects of an extract from Cladonia rangiformis. HOFFM Biol Pharm Bull 2002; 25:10-3.

Karunaratne V, Bombuwela K, Kathirgamanathar S, Thadhani VM. Lichens: a chemically important biota. J Natn Sci Foundantion Sri Lanka 2005; 33:169-86.

Oran S, Sahin S, Sahinturk P, Oztürk S, Demir C. Antioxidant and Antimicrobial Potential, and HPLC Analysis of Stictic and Usnic Acids of Three Usnea Species from Uludag Mountain (Bursa, Turkey). Iran J Pharm Res 2016; 15:527-35.

Siegel R, Naishadham D, Jemal A. Cancer statistics. CA Cancer Journal for Clinicians 2013; 63:11-30.

Sosa V, Moliné T, Somoza R, Paciucci R, Kondoh H, LLeonart ME. Oxidative stress and cancer: an overview. Ageing Res Rev 2013; 12(1):376-90.

Martinez-Useros J, Li W, Cabeza-Morales M, Garcia-Foncillas J. Oxidative Stress: A New Target for Pancreatic Cancer Prognosis and Treatment. J Clin Med 2017; 6(3):E29.

Hecht F, Pessoa CF, Gentile LB, Rosenthal D, Carvalho DP, Fortunato RS. The role of oxidative stress on breast cancer development and therapy. Tumour Biol 2016; 37(4):4281-91.

Ren MR, Hur JS, Kim JY, Park KW, Park SC, Seong CN, et al. Anti-proliferative effects of Lethariella zahlbruckneri extracts in human HT-29 human colon cancer cells. Food Chem Toxicol 2009; 47:2157-62.

Manojlović N, Ranković B, Kosanić M, Vasiljević P, Stanojković T. Chemical composition of three Parmelia lichens and antioxidant, antimicrobial and cytotoxic activities of some their major metabolites. Phytomedicine 2012; 19:1166-72.

Brandí£o LF, Alcantara GB, Matos Mde F, Bogo D, Freitas Ddos S, Oyama NM, et al. Cytotoxic evaluation of phenolic compounds from lichens against melanoma cells. Chem Pharm Bull 2013; 61:176-83.

Su ZQ, Liu YH, Guo HZ, Sun CY, Xie JH, Li YC, et al. Effect-enhancing and toxicity-reducing activity of usnic acid in ascitic tumor-bearing mice treated with bleomycin. Int Immunopharmacol. 2017; 46:146-55.

Bézivin C, Tomasi S, Lohézic-Le Dévéhat F, Boustie J. Cytotoxic activity of some lichen extracts on murine and human cancer cell lines. Phytomedicine 2003; 10:499-503.

Baćkorová M, Jendželovskí½ R, Kello M, Baćkor M, Mikeš J, Fedoroćko P. Lichen secondary metabolites are responsible for induction of apoptosis in HT-29 and A2780 human cancer cell lines. Toxicol In Vitro 2012; 26:462-8.

Subedi A, Amatya MP, Shrestha TM, Mishra SK, Pokhrel BM. Antioxidant and antibacterial activity of methanolic extract of Machilus odoratıssıma. KUSET 2012; 8:73-80.

Smith CW, Aptroot A, Coppins BJ, Fletcher A, Gilbert OL, James PW, et al. The lichens of Great Britain and Ireland. The British Lichen Society, London, 2009, pp.309-38.

Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1963; 61:882-8.

Ledwozyw A, Michalak J, StepieÅ„ A, KadzioÅ‚ka A. The relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis. Clin Chim Acta 1986; 155(3):275-83.

Aebi H. Catalase in vitro. Methods Enzymol 1984; 105:121–6.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193(1):265-75.

Bauer AW, Kirby WMN, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized simple disc method. Am J Clin Pathol 1966; 45:493.

Fridlender M, Kapulnik Y, Koltai H. Plant derived substances with anti-cancer activity: from folklore to practice. Front Plant Sci. 2015; 6:799

Acikgoz B, Karalti I, Ersoz M, Coskun ZM, Cobanoglu G, Sesal C. Screening of antimicrobial activity and cytotoxic effects of two Cladonia species. Z Naturforsch C 2013; 68:191-7.

Mitrović T, Stamenković S, Cvetković V, Tošić S, Stanković M, Radojević I, et al. Antioxidant, antimicrobial and antiproliferative activities of five lichen species. Int J Mol Sci 2011; 12:5428-48.

Ranković BR, Kosanić MM, Stanojković TP. Antioxidant, antimicrobial and anticancer activity of the lichens Cladonia furcata, Lecanora atra and Lecanora muralis. BMC Complement Altern Med 2011; 11:97-104.

Singh N, Nambiar D, Kale RK, Singh RP. Usnic acid inhibits growth and induces cell cycle arrest and apoptosis in human lung carcinoma A549 cells. Nutr Cancer 2013; 65:36-43.

Kosanić MM, Ranković BR, Stanojković TP. Antioxidant, antimicrobial and anticancer activities of three Parmelia species. J Sci Food Agric 2012; 92:1909-16.

Kosanić M, Ranković B, Stanojković T. Antioxidant, antimicrobial, and anticancer activity of 3 Umbilicaria species. J Food Sci 2012; 77:T20-5.

Basile A, Rigano D, Loppi S, Di Santi A, Nebbioso A, Sorba Sergio, et al. Antiproliferative, antibacterial and antifungal activity of the lichen Xanthoria parietina and its secondary metabolite parietin. Int J Mol Sci 2015; 16:7861-75.

Levkoff LH, Marshall GP 2nd, Ross HH, Caldeira M, Reynolds BA, Cakiroglu M, et al. Bromodeoxyuridine inhibits cancer cell proliferation in vitro and in vivo. Neoplasia. 2008; 10(8):804-16.

Stoimenov I, Helleday T. PCNA on the crossroad of cancer. Biochem Soc Trans. 2009; 37(Pt 3):605-13.

Romagni JG, Dayan FE. Structural diversity of lichen metabolites and their potential use. In: Advances in microbial toxin research and its biotechnological exploitation. Upadhyay RK. (ed), Kluwer Academic/Plenum Publishers, New York, 2002, pp.151-70.

Mayer M, O'Neill MA, Murray KE, Santos-Magalhaes NS, Carneiro-Leao AM, Thompson AM, et al. Usnic acid: a non-genotoxic compound with anticancer properties. Anti Canc Drugs 2005; 16:805-9.

Brisdelli F, Perilli M, Sellitri D, Piovano M, Garbarino JA, Nicoletti M, et al. Cytotoxic activity and antioxidant capacity of purified lichen metabolites: an in vitro study. Phytother Res. 2013; 27(3):431-7.

Yilmaz M, Ozdemir Turk A, Tay T, Kivanc M. The antimicrobial activity of extracts of the lichen Cladonia foliacea and its (–)-usnic acid, atranorin, and fumarprotocetraric acid constituents. Z Naturforsch C 2004; 59:249-54.

Ingolfsdottir K. Molecules of interest – usnic acid. Phytochemistry 2002; 61:729-36.

Cobanoglu G, Sesal C, Gokmen B, Cakar S. Evaluation of the antimicrobial properties of some lichens. South West J Hortic Biol Environ 2010; 1:153-8.

Ranković B, Mišic M, Sukdolak S. Antimicrobial activity of extracts of the lichens Cladonia furcata, Parmelia caperata, Parmelia pertusa, Hypogymnia physodes and Umbilicaria polyphylla. Biologia 2009; 64:53-8.