inhibition of protein kinase c αβii and activation of c-jun nh2-terminal kinase mediate glycyrrhetinic acid induced apoptosis

inhibition of protein kinase c αβii and activation of c-jun nh2-terminal kinase mediate glycyrrhetinic acid induced apoptosis 作文 手机作文网 Inhibition of protein kinase C αβII and activation of c-Jun NH2-terminal kinase mediate glycyrrhetinic acid induced apoptosis Inhibition of protein kinase C a/bII and activation of c-JunNH2-terminal kinase mediate glycyrrhetinic acid induced apoptosisin non-small cell lung cancer NCI-H460 cellsJunho Songa,?, Hyun-suk Koa,?, Eun Jung Sohna,?, Bonglee Kima, Jung Hyo Kimb, Hee Jeong Kimc,Chulwoo Kima, Jai-eun Kimd, Sung-Hoon Kima,?aCancer Preventive Material Development Center, College of Oriental Medicine, Kyung Hee University, 1 Hoegi-dong, Research Dongdaemun-gu, Seoul 130-701, South KoreabChosun Nursing College, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, South KoreacGraduate School of East-West Medical Science, Kyung Hee University, Yongin 446-701, South KoreadDepartment of Oriental Pathology, College of Korean Medicine, Dongguk University, 814-9 Ilsandong-gu, Goyang, Geyonggi, South Koreaa r t i c l ei n f oArticle history:Received 30 September 2013Revised 24 December 2013Accepted 24 December 2013Available online 3 January 2014Keywords:Glycyrrhetinic acidCaspasePAPRProtein kinase CJNKNCI-H460a b s t r a c tThough glycyrrhetinic acid (GA) from Glycyrrhiza glabra known to exert antioxidant, antifilarial,hepatoprotective, anti-inflammatory and was anti-tumor effects, the antitumor mechanism of GA was notclearly elucidated in non-small cell lung cancer cells (NSCLCCs). Thus, in the present study, the underly-ing apoptotic mechanism of GA was examined in NCI-H460 NSCLCCs. GA significantly suppressed the via-bility of NCI-H460 and A549 non-small lung cancer cells. Also, GA significantly increased the sub G1population by cell cycle analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUN-EL) positive cells in a concentration dependent manner in NCI-H460 non-small lung cancer cells. Consis-tently, GA cleaved poly (ADP-ribosyl) polymerase (PARP), caspase 9/3, attenuated the expression ofBcl-XL, Bcl-2, Cyclin D1 and Cyclin E in NCI-H460 cells. Interestingly, GA attenuated the phosphorylationof protein kinase C (PKC) a/bII and extracellular activated protein kinase (ERK) as well as activated thephosphorylation of PKC d and c-Jun NH2-terminal kinase in NCI-H460 cells. Conversely, PKC promoterphorbol 12-myristate 13-acetate (PMA) and JNK inhibitor SP600125 reversed the cleavages of caspase3 and PARP induced by GA in NCI-H460 cells. Overall, our findings suggest that GA induces inhibition of PKC a/bII and activation of JNK in NCI-H460 non-small lung cancer apoptosisvia cells as a potent anti-cancer candidate for lung cancer treatment.? 2013 Elsevier Ltd. All rights reserved.Non-small-cell lung cancer (NSCLC) is one of the most commonmalignant diseases worldwide. The most common types of NSCLCare adenocarcinoma (ADC) and squamous cell carcinoma (SCC).1,2Though platinum-based chemotherapy including cisplatin or car-boplatin has been applied to over half of the patients with NSCLCfor years, the 5-year relative survival rate is approximately 15.7–17.5% and most patients eventually progressed and passed awaydue to their malignant disease during the last decade.3,4Sincethe side effects of cisplatin are known severe with anemia, neutro-penia, neurotoxicity and nausea,5recently herbal drugs or naturalcompounds are attractive for the prevention or treatment forNSCLCs alone or by combination therapy with cisplatin.6–8Ant-roquinonol induced apoptosis in A549 作文 手机作文网 作文 手机作文网 cells via inhibition of PI3Kand mTOR signaling9carvacrol, the predominant monoterpene inmany essential oils, induced apoptosis and inhibited the growthof A549 cells10and curcumin inhibited migration and invasion ofA549 cells11and also induced apoptosis in NCI-H 460 cells.12In this regard, glycyrrhetinic acid (GA), an agylcone of glycyrrhi-zin contained in aqueous extract of licorice or Glycyrrhiza glabra,was known to have a variety of biological activities such asanti-allergic,13hepatoprotective,14,15anti-inflammatory,16–18anti-microbial19and antitumor effects.20,21Among those biologicalactivities, though GA was reported to have antitumor activity inseveral cancers such as liver cancer,22breast cancer,23prostatecancer24,25and pancreatic cancer,26the underlying mechanism ofGA in lung cancer still remains unclear until now. Thus, in thecurrent study, the molecular mechanism of GA in NSCLCs wasinvestigated in association with protein kinase C a/bII and c-JunNH2-terminal kinase pathways.To assess the antitumor activity of GA (Fig. 1A) in NSCLCs such asA549andNCI-H460,XTTcolorimetricassaywasperformed.Asshownin Figure 1B, GA showed significant cytotoxicity in A549 and NCI-H460 cells with IC50of approximately 78 and 62 lg/ml, respectively.To assess the effect of GA on the apoptosis related proteins,Western blotting was performed. GA activated cleaved PAPR andcaspase 9/3 (Fig. 2A) and attenuated the expression of Bcl-XL,Bcl-2, Cyclin D1 and Cyclin E (Fig. 2B) in NCI-H460 cells.0960-894X/$ - see front matter ? 2013 Elsevier Ltd. All rights reserved.: +82 2 961 9233; fax: +82 2 964 1074.E-mail address: sungkim7@khu.ac.kr (S.-H. Kim).?Equally authors.Bioorganic & Medicinal Chemistry Letters 24 (2014) 1188–1191Contents contributed lists available at ScienceDirectBioorganic & Medicinal Chemistry Lettersjournal homepage: www.elsevier.com/locate/bmcl Furthermore, apoptotic cells can be identified by DNA content fre-quency histograms as cells with fractional sub-G1 DNA content.27TUNEL assay is a common method for detecting DNA fragmenta-tion resulting from apoptotic signaling cascades.28GA increasedthe sub G1 population to 2.8%, 11.3% and 40.1% at the concentra-tions of 12.5, 25 and 50 lM compared to untreated control (2.2%)in NCI-H460 cells by cell cycle analysis (Fig. 2C). Consistently, GAenhanced green color TUNEL positive cells compared to untreatedcontrol by TUNEL assay in NCI-H460 cells (Fig. 2D). We also con-firmed that GA induces apoptosis in A549 cells by Western blot(Fig. 3A) and FACS assay (Fig. 3B). Thus, our results demonstratethat GA induces apoptosis in NSCLC.GA suppressed the phosphorylation of protein kinase C (PKC) a/bII and activated the phosphorylation of PKC d (Fig. 4A), and alsoreduced the phosphorylation of extracellular activated protein ki-nase (ERK) and activated the phosphorylation of c-Jun NH2-termi-nal kinase (Fig. 4B) in NCI-H460 cells. To confirm the critical role ofJNK and PKC a/bII in GA induced apoptosis in NCI-H460 cells, theeffect of JNK inhibitor SP600125 and PKC promoter phorbol 12-myristate 13-acetate (PMA) on GA induced apoptosis was evalu-ated in NCI-H460 cells. As shown in Fig. 4C, JNK inhibitorSP600125 and PKC promoter PMA reversed the cleavages of cas-pase 3 and PARP and inhibition of PKC a/bII induced by GA inNCI-H460 cells. Furthermore, FACS assay confirmed that subG1portion induced by GA was reduced by PMA or SP600125 com-pared with GA treated group alone (Fig. 4D).We focused on the underlying apoptotic mechanism of GA inNCI-H460 non-small lung cancer cells. In the current study, GAPARP cleaved caspase-3 β-actinC 12.5 25 50 (μM) C 12.5 25 50 (μM) Bcl-xLBcl-2 Cyclin D1Cyclin E caspase-9 β-actinGA GA (B)control12.5 μM 25 μM 50 μM 作文 手机作文网 作文 手机作文网 (A)012.52550050100G2SG1Sub-G1GA (μμM)DNA contents (% of Control)(D)Con50 μM DAPI FITCMerge(C)GA Figure 2. Effect of GA on inducing apoptosis in NCI-H460 cells. (A and B) of GA on apoptotic and anti-apoptotic proteins in NCI-H460 cells after culture for Effect by Westernblotting. (C) Effect of GA on sub G1 population in NCI-H460 cells by cell cycle analysis. Graphs represent cell percentages at each phase of cell cycle (D) Effect of GA on TUNELpositive cells in NCI-H460 cells. NCI-H460 cells were exposed to 50 lM of GA for 24 h and TUNEL positive cells were observed using a fluorescence microscopy (OlympusIX70, San Diego, USA).(A)(B)MW : 470.68Glycyrrhetinic acid?020406080100020406080100120------------------------------------------------NCI-H460A549Concentration (μM)Cell viability (%)Figure 1. Chemical structure of glycyrrhetinic acid (GA) and its cytotoxicity inNSCLCs. (A) Chemical structure of GA; (B) cytotoxicity of GA in NSCLCs includingA549 and NCI-H460 cells. The cytotoxicity of GA was measured using XTTcolorimetric assay.J. Song et al./Bioorg. Med. Chem. Lett. 24 (2014) 1188–11911189 significantly suppressed the viability of NCI-H460 and A549 non-small cell lung cancer cells (NSCLCCs), implying antitumor poten-tial of GA in lung cancer cells. To assess whether the cytotoxicityof GA is due to apoptosis induction, cell cycle analysis and TUNELassay was performed. Here GA significantly increased the sub G1population by cell cycle analysis and terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) positive cells in a con-centration dependent manner in NCI-H460 cells, indicating thecytotoxicity of GA is induced by apoptosis in NCI-H460 cells.Caspase family, aspartate-specific cysteine are in-volved in the regulation of apoptosis.29Activation of caspase-3and proteases, following cleavage of its substrates such as PARP and laminA are the hallmarks of apoptosis.30In mitochondria-dependentapoptosis pathway, caspase-9 with cytochrome c and -1 formthe apoptosome in which caspase-9 is activated to trigger cleavageof other Apaf caspases including caspase-3.31We found that GA cleavedPARP, caspase 9 and caspase3 in NCI-H460 cells, implying the cas-pase dependent apoptosis of GA.Apoptosis signaling is regulated by various pro- and anti-apop-totic proteins. Bcl-2 family proteins is localized in mitochondriaand control caspase activation.32,33GA attenuated the expressionof anti-apoptotic proteins such as Bcl-XL, Bcl-2, Cyclin D1 and Cy-clin E in NCI-H460 cells, indicating GA regulates survival genesleading to apoptosis induction.Protein kinase C (PKC) family members are involved in pro-sur-vival or pro-apoptotic activity depending on the specific isoform.34It is well documented that among them, PKC d is a ubiquitously ex-pressed isoform that serve as a pro-apoptotic mediator35,36, whileControlGA 50 μM 050050100G2SG1Sub-G1GA (50 μM)DNA contents (% of Control)C 12.5 25 50 (μM) PARP β-actinGA (B)(A) Figure 3. Effect of GA on inducing apoptosis in A549 cells. (A) Effect of GA onapoptotic protein, PARP in A549 cells by Western blotting. (B) Effect of GA on subG1 population in A549 cells by cell cycle analysis. Graphs represent cell percentagesat each phase of cell cycle.p-PKC α/βp-PKC δβ-actinC 12.5 25 50 (μM) C 12.5 25 50 (μM) p-ERKp-JNKβ-actinERKJNK(A) GA GA (B) -+ -+ -+ GA (50 μM) --+ + --PMA (50 ng/ml) p-PKC α/β ----+ + SP600125 (30 μM) β-actincleaved caspase-3 PARP p-JNKJNK(C)controlGA 50 μM PMA 50 ng/mlPMA 50 ng/ml+ GA 50 μM SP600125 30 μM SP600125 30 μM + GA 50 μM (D)050100G2SG1Sub-G1GA (50 μM) - + - + - +PMA (50 ng/ml) - - + + - -SP600125 (30 μM) - - - - + +DNA contents (% of Control)Figure 4. Effect of PKC promoter PMA and JNK inhibitor SP600125 on the apoptosis 作文 手机作文网 作文 手机作文网 induced by GA in NCI-H460 cells. (A and B) Effect of GA on PKC, ERK and JNK molecules inNCI-H460 cells. NCI-H460 cells were exposed to GA (50 lM) for 24 h and Western blot was carried out with indicated antibodies. (C and D) NCI-H460 cells were exposed toGA (50 lM) with PMA or SP600125 for 24 h. Western blotting (C) and FACS (D) were performed. Graphs represent cell percentages at each phase of cell cycle.1190J. Song et al./Bioorg. Med. Chem. Lett. 24 (2014) 1188–1191 PKC a/bII promotes survival in the cells.37,38There is evidence thatProtein kinase C d activates the MAP kinase /ERK kinase pathway.39Interestingly, GA attenuated the phosphorylation of protein kinaseC (PKC) a/bII and extracellular activated protein kinase(ERK) aswell as activated the phosphorylation of PKC d and c-Jun NH2-ter-minalkinaseinNCI-H460cells.Conversely,JNKinhibitorSP600125 and PKC promoter PMA reversed the cleavages of cas-pase 3 and PARP and inhibition of PKC a/bII induced by GA -H460 cells. Overall, our findings suggest that GA induces apop-tosis via inhibition inNCI of PKC a/bII and activation of JNK in NCI-H460non-small lung cancer cells as a potent anticancer candidate forlung cancer treatment.AcknowledgmentsThis work was supported by the Korea Science and EngineeringFoundation (KOSEF) Grant funded by the Korea Government(MEST) (No. 2012-0005755). The authors declare no competinginterests.Supplementary dataSupplementary data associated with this article can be found, inthe online version, at , M. A. Clin. Cancer Res. 2004, 10, 4210s.2. Klastersky, J.; Awada, A. Crit. Rev. Oncol. Hematol. 2011, 81, 49.3. Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D. CA Cancer J.Clin. 2011, 61, 69.4. Wao, H.; Mhaskar, R.; Kumar, A.; Miladinovic, B.; Djulbegovic, B. Syst. Rev. 2013,2, 10. , A.; Trojan, A.; Burnand, B.; Giannelli, M. Lung Cancer 2008, 59, 1.6. Chen, S.; Flower, A.; Ritchie, A.; Liu, J.; Molassiotis, A.; Yu, H.; Lewith, G. LungCancer 2010, 68, 137.7. Zhang, Y.; Zeng, F.; Liu, X.; Li, Y.; Zhou, J.; Huang, Y.; Wang, Y.; Zhou, S.; Zhu, W.;Shu, E.; Zhou, G.; Chen, G. Xenobiotica. 2011, 41, 593.8. Grozio, A.; Paleari, L.; Catassi, A.; Servent, D.; Cilli, M.; Piccardi, F.; Paganuzzi,M.; Cesario, A.; Granone, P.; Mourier, G.; Russo, P. Int. J. Cancer 2008, 122, 1911.9. Kumar, V. B.; Yuan, T. C.; Liou, J. W.; Yang, C. J.; Sung, P. J.; Weng, C. F. Mutat.Res. 2011, 707, 42.10. Koparal, A. T.; Zeytinoglu, M. Cytotechnology 2003, 43, 149.11. Lin, S. S.; Lai, K. C.; Hsu, S. C.; Yang, J. S.; Kuo, C. L.; Lin, J. P.; Ma, Y. S.; Wu, C. C.;Chung, J. G. Cancer Lett. 2009, 285, 127.12. Wu, S. H.; Hang, L. W.; Yang, J. S.; Chen, H. Y.; Lin, H. Y.; Chiang, J. H.; Lu, C. C.;Yang, J. L.; Lai, T. Y.; Ko, Y. C.; Chung, J. G. Anticancer Res. 2010, 30, 2125.13. Akasaka, Y.; Yoshida, T.; Tsukahara, M.; Hatta, A.; Inoue, H. Eur. J. Pharmacol.2011, 670, 175.14. Chen, S.; Zou, L.; Li, L.; Wu, T. PLoS ONE 2013, 8, e53662.15. Tian, Q.; Wang, X.; Wang, W.; Zhang, C.; Yuan, Z.; Chen, X. J. Control Release.2011, 152, e237.16. Gui, M.; Li, Y.; Wu, T.; Huang, Y.; Zhang, H. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi2012, 28, 915.17. Ishida, T.; Mizushina, Y.; Yagi, S.; Irino, Y.; Nishiumi, S.; Miki, I.; Kondo, Y.;Mizuno, S.; Yoshida, H.; Azuma, T.; Yoshida, M. Evid. Based Complement. Altern.Med. 2012, 2012, 650514.18. Kao, T. C.; Shyu, M. H.; Yen, G. C. J. Agric. Food Chem. 2010, 58, 8623.19. Gaware, R.; Khunt, R.; Czollner, L.; Stanetty, C.; Da Cunha, T.; Kratschmar, D. V.;Odermatt, A.; Kosma, P.; Jordis, U.; Classen-Houben, D. Bioorg. Med. Chem.2011, 19, 1866.20. Csuk, R.; Schwarz, S.; Siewert, B.; Kluge, R.; Strohl, D. Arch. Pharm. (Weinheim)2012, 345, 223.21. Gao, Z.; Kang, X.; Hu, J.; Ju, Y.; Xu, C. Cytotechnology 2012, 64, 421.22. Lai, Y.; Shen, L.; Zhang, Z.; Liu, W.; Zhang, Y.; Ji, H.; 作文 手机作文网 作文 手机作文网 Tian, J. Bioorg. Med. Chem.Lett. 2010, 20, 6416.23. Sharma, G.; Kar, S.; Palit, S.; Das, P. K. J. Cell. Physiol. 2012, 227, 1923.24. Shetty, A. V.; Thirugnanam, S.; Dakshinamoorthy, G.; Samykutty, A.; Zheng, G.;Chen, A.; Bosland, M. C.; Kajdacsy-Balla, A.; Gnanasekar, M. Int. J. Oncol. 2012,39, 635.25. Hawthorne, S.; Gallagher, S. J. Pharm. Pharmacol. 2008, 60, 661.26. Jutooru, I.; Chadalapaka, G.; Chintharlapalli, S.; Papineni, S.; Safe, S. Mol.Carcinog. 2009, 48, 692.27. Kajstura, M.; Halicka, H. D.; Pryjma, J.; Darzynkiewicz, Z. Cytometry A 2007, 71,125.28. Negoescu, A.; Guillermet, C.; Lorimier, P.; Brambilla, E.; Labat-Moleur, F.Biomed. Pharmacother. 1998, 52, 252.29. Stennicke, H. R.; Salvesen, G. S. Biochim. Biophys. Acta 1998, 1387, 17.30. Hengartner, M. O. Nature 2000, 407, 770.31. Li, P.; Nijhawan, D.; Budihardjo, I.; Srinivasula, S. M.; Ahmad, M.; Alnemri, E. S.;Wang, X. Portt, L.; Norman, G.; Clapp, C.; Greenwood, M.; Greenwood, M. T. Cell 1997, 91, 479.32. Biochim.Biophys. Acta 2011, 1813, 238.33. Zamzami, N.; Brenner, C.; Marzo, I.; Susin, S. A.; Kroemer, G. Oncogene 1998, 16,2265.34. Webb, B. L.; Hirst, S. J.; Giembycz, M. A. Br. J. Pharmacol. 2000, 130, 1433.35. Lu, P. H.; Yu, C. C.; Chiang, P. C.; Chen, Y. C.; Ho, Y. F.; Kung, F. L.; Guh, J. H. J.Urol. 2011, 186, 2434.36. Haughian, J. M.; Jackson, T. A.; Koterwas, D. M.; Bradford, A. P. Endocr. Relat.Cancer 2006, 13, 1251.37. Jung, S. Y.; Kim, O. B.; Kang, H. K.; Jang da, H.; Min, B. M.; Yu, F. H. Exp. Cell Res.2013, 319, 153.38. Aaltonen, V.; Peltonen, J. Anticancer Res. 2010, 30, 3995.39. Ueda, Y.; Hirai, S.; Osada, S.; Suzuki, A.; Mizuno, K.; Ohno, S. J. Biol. Chem. 1996,271, 23512.J. Song et al./Bioorg. Med. Chem. Lett. 24 (2014) 1188–11911191 作文 手机作文网