Evaluation of ameliorative potential of isolated flavonol fractions from Thuja occidentalis in lung cancer cells and in Benzo(a)pyrene induced lung toxicity in mice


Evaluation of ameliorative potential of isolated flavonol fractions from Thuja occidentalis in lung cancer cells and in Benzo(a)pyrene induced lung toxicity in mice


Avinaba Mukherjee1, Sourav Sikdar1 and Anisur Rahman Khuda-Bukhsh1*International Journal of Traditional and Complementary Medicine

1Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, West Bengal, India.

Review method: Single-blind; Peer reviewer comments: 2.


Relative anticancer potentials of six flavonol fractions (F1-F6) isolated from Thuja occidentalis extract were first evaluated against lung cancer cells A549 in vitro, and fraction- F4 showing the maximum protective ability, was then tested in mice intoxicated with Benzo(a)pyrene (BaP), a known carcinogen with major effect on lung, to learn if this could also have ameliorative action against lung toxicity and tissue damage in mice in vivo. Chemical nature of F1-F6 fractions was confirmed with specific flavonol confirmatory test and mass spectral analysis; all fractions were tested for their possible anticancer effects against A549 cells. Results suggested that fraction 4 (F4) had the strongest anticancer effect. When treated to BaP intoxicated mice, F4 induced recovery of damaged lung tissue, presumably through inhibition of ROS generation, and enhanced production of major antioxidant molecules, that in turn blocked PI3K-activated expressions of Akt. Decrease in Bcl2/Bax ratio, over-expression of p53 gene and activation of caspase 3 were observed in tissues of F4 treated mice, further confirming apoptotic cell death as its major target. The F4 fraction of Thuja occidentalis extract showed remarkable apoptotic potential against lung cancer cells and ameliorative ability against BaP induced lung toxicity.

Flavonols ameliorate Benzo[a]pyrene-induced lung toxicity


Keywords: Flavonol; benzo[a]pyrene toxicity; lung toxicity; PI3K/Akt pathway; reactive oxygen species (ROS), Thuja occidentalis.

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How to cite this article:
Avinaba Mukherjee, Sourav Sikdar, Anisur Rahman Khuda-Bukhsh. Evaluation of ameliorative potential of isolated flavonol fractions from Thuja occidentalis in lung cancer cells and in Benzo(a)pyrene induced lung toxicity in mice. International Journal of Traditional and Complementary Medicine 2016, 1:1. DOI:10.28933/mukherjee-ijtcm-2016


References:
1. Rebecca L, Siegel MPH, Kimberly D, Miller MPH, Ahmedin-Jemal DVM (2016) Cancer statistics. CA: A Cancer Journal for Clinicians. 66: 7-30.
2. Asokkumar S, Naveenkumar C, Raghunandhakumar S, Kamaraj S, Anandakumar P, Jagan S, Devaki T (2012). Antiproliferative and antioxidant potential of beta-ionone against benzo(a)pyrene-induced lung carcinogenesis in Swiss albino mice. Mol. Cell. Biochem. 363: 335-345.
3. Sikdar S, Mukherjee A, Ghosh S, Khuda-Bukhsh AR (2014).Condurango glycoside-rich components stimulate DNA damage-induced cell cycle arrest and ROS-mediated caspase-3 dependent apoptosis through inhibition of cell-proliferation in lung cancer, in vitro and in vivo. Environ. Toxicol. Pharmacol. 37: 300-314.
4. Kazerouni N, Sinha R, Hsu CH, Greenberg A, Rothman N (2001). Analysis of 200 food items for benzo[a]pyrene and estimation of its intake in an epidemiologic study. Food. Chem. Toxicol. 39: 423-436.
5. Kim HS, Lee BM (1997). Inhibition of benzo[a]pyrene-DNA adduct formation by Aloe barbadensis Miller. Carcinogenesis.18: 771-776.
6. Barnes SL, Singletary KW, Frey R (2000). Ethanol and acetaldehyde enhance benzo[a]pyrene-DNA adduct formation in human mammary epithelial cells. Carcinogenesis. 21: 2123-2128.
7. Briede JJ, Godschalk RW, Emans MT, De-Kok TM, Van-Agen E, Van-Maanen J, Van-Schooten FJ, Kleinjans JC (2004). In vitro and in vivo studies on oxygen free radical and DNA adduct formation in rat lung and liver during benzo[a]pyrene metabolism. Free. Radic. Res. 38: 995-1002.
8. Busch C, Burkard M, Leischner C, Lauer UM, Frank J, Venturelli S (2015). Epigenetic activities of flavonoids in the prevention and treatment of cancer. Clin. Epigenetics.7: 64.
9. Cui Y, Morgenstern H, Greenland S, Tashkin DP, Mao JT, Cai L, Cozen W, Mack TM, Lu QY, Zhang ZF (2008). Dietary flavonoid intake and lung cancer-a population-based case-control study. Cancer. 112: 2241-2248.
10. Tsiri D, Graikou K, Poblocka-Olech L, Krauze-Baranowska M, Spyropoulos C, Chinou I (2009). Chemosystematic value of the essential oil composition of Thuja species cultivated in Poland-antimicrobial activity. Molecules. 14: 4707-4715.
11. Naser B, Bodinet C, Tegtmeier M, Lindequist U (2005). Thujaoccidentalis (Arbor vitae): A Review of its Pharmaceutical, Pharmacological and Clinical Properties. Evid. Based. Complement. Alternat. Med. 2: 69-78.
12. Mukherjee A, Sikdar S, Bishayee K, Boujedaini N, Khuda-Bukhsh AR (2014). Flavonol isolated from ethanolic leaf extract of Thuja occidentalis arrests the cell cycle at G2-M and induces ROS-independent apoptosis in A549 cells, targeting nuclear DNA. Cell. Prolif. 47: 56-71.
13. Mukherjee A, Sikdar S, Bishayee K, Paul A, Ghosh S, Boujedaini N, Khuda-Bukhsh AR (2012) Ethanolic extract of Thujaoccidentalis blocks proliferation of A549 cells and induces apoptosis in vitro. Zhong Xi Yi Jie He XueBao. 10: 1451-1459.
14. Roychoudhury S, Mondal NK, Mukherjee S, Dutta A, Siddique S, Ray MR (2012). Activation of protein kinase B (PKB/Akt) and risk of lung cancer among rural women in India who cook with biomass fuel. Toxicol. Appl. Pharmacol. 259: 45-53.
15. Zhao P, Fu J, Yao B, Song Y, Mi L, Li Z, Shang L, Hao W, Zhou Z (2012). In vitro malignant transformation of human bronchial epithelial cells induced by benzo(a)pyrene. Toxicol. In Vitro. 26: 362-368.
16. Dubey SK, Batra A (2009). Antioxidant Activities of Thujaoccidentalis Linn. Asian. J .Pharm. Clin. Res. 2: 73-76.
17. Olusola A, Olutayo O, Michael A, Olakunle F, Edah AO (2011). Elemental analysis and Anti-microbial potentials of the leaf extract of Cassia arereh Del. Int. Res. J. Pharm. Pharmacol. 1: 188-193.
18. Anandakumar P, Kamaraj S, Ramakrishnan G, Jagan S, Devaki T (2009). Chemopreventive task of capsaicin against benzo(a)pyrene-induced lung cancer in Swiss albino mice. Basic. Clin. Pharmacol. Toxicol. 104: 360-365.
19. Fuss IJ, Kanof ME, Smith PD, Zola H (2009). Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr. Protoc. Immunol. doi:10.1002/0471142735.im0701s85.
20. Chakraborty D, Mukherjee A, Sikdar S, Paul A, Ghosh S, Khuda-Bukhsh AR (2012) [6]-Gingerol isolated from ginger attenuates sodium arsenite induced oxidative stress and plays a corrective role in improving insulin signaling in mice. Toxicol. Lett. 210: 34-43.
21. Rahman I, Kode A, Biswas SK (2006). Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc. 1: 3159-3165.
22. Goh FY, Upton N, Guan S, Cheng C, Shanmugam MK, Sethi G, Leung BP (2012). Wong WS. Fisetin, a bioactive flavonol, attenuates allergic airway inflammation through negative regulation of NF-κB. Eur. J. Pharmacol. 679: 109-116.
23. Kundu S, Sengupta S, Chatterjee S, Mitra S, Bhattacharyya A (2009). Cadmium induces lung inflammation independent of lung cell proliferation: a molecular approach. J. Inflamm. (Lond). 6: 19.
24. Efferth T, Giaisi M, Merling A, Krammer PH, Li-Weber M (2007). Artesunate induces ROS-mediated apoptosis in doxorubicin-resistant T leukemia cells. PLoS One. 2: e693.
25. Liu C, Liu H, Li Y, Wu Z, Zhu Y, Wang T, Gao AC, Chen J, Zhou Q (2001). Intracellular glutathione content influences the sensitivity of lung cancer cell lines to methylseleninic acid. Mol. Carcinog. 51: 303-314.
26. Sarkar M, Chaudhuri K (2004). Association of adherence and motility in interleukin 8 induction in human intestinal epithelial cells by Vibrio cholerae. Microbes. Infect. 6: 676-685.
27. Schneider CA, Rasbandws Eliceiri KW (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods. 9: 671-675.
28. Ganguly A, Basu S, Chakraborty P, Chatterjee S, Sarkar A, Chatterjee M, Choudhuri SK (2010). Targeting mitochondrial cell death pathway to overcome drug resistance with a newly developed iron chelate. PLoS One. 5: e11253.
29. Benzie IF, Szeto YT, Strain JJ, Tomlinson B (1999). Consumption of green tea causes rapid increase in plasma antioxidant power in humans. Nutr. Cancer. 34: 3-7.
30. Tietze F (1969). Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 27: 502-522.
31. Weydert CJ, Cullen JJ (2010). Measurement of superoxide dismutase, catalase and glutathione peroxidase in cultured cells and tissue. Nat. Protoc. 5: 51-66.
32. Sambrook J, Russell DW (2001). Molecular Cloning, Cold Spring Harbor Laboratory Press, New York. 2001; 3rd ed.
33. Tsuji G, Takahara M, Uchi H, Takeuchi S, Mitoma C, Moroi Y, Furue M (2011). An environmental contaminant, benzo(a)pyrene, induces oxidative stress-mediated interleukin-8 production in human keratinocytes via the aryl hydrocarbon receptor signaling pathway. J. Dermatol. Sci. 62: 42-49.
34. Wu H, Che X, Zheng Q, Wu A, Pan K, Shao A, Wu Q, Zhang J, Hong Y (2014). Caspases: a molecular switch node in the crosstalk between autophagy and apoptosis. Int J Biol Sci. 10: 1072-1083.
35. Xu N, Lao Y, Zhang Y, Gillespie DA (2012). Akt: a double-edged sword in cell proliferation and genome stability. J. Oncol.doi: 10.1155/2012/951724.
36. Chin YR, Toker A (2009). Function of Akt/PKB signaling to cell motility, invasion and the tumorstroma in cancer. Cell Signal. 21: 470-476.
37. Fresno Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, González-Barón M (2004). PI3K/Aktsignalling pathway and cancer. Cancer Treat. Rev. 30: 193-204.
38. Reagan-Shaw S, Nihal M, Ahmad N (2008). Dose translation from animal to human studies revisited. FASEB. J. 22: 659-661.