The cathelicidin-BF Lys16 mutant Cbf-K16 selectively inhibits non-small cell lung cancer proliferation in vitro

Tian,Yuwei; Wang,Hui; Li,Bing; Ke,Mengyun; Wang,Jing; Dou,Jie; Zhou,Changlin

doi:10.3892/or.2013.2693

The cathelicidin-BF Lys16 mutant Cbf-K16 selectively inhibits non-small cell lung cancer proliferation in vitro

Authors:
- Yuwei Tian
- Hui Wang
- Bing Li
- Mengyun Ke
- Jing Wang
- Jie Dou
- Changlin Zhou
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Affiliations: State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu 210009, P.R. China
Published online on: August 23, 2013 https://doi.org/10.3892/or.2013.2693
Pages: 2502-2510

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Abstract

The 30-amino acid antimicrobial peptide Cbf-K16 is a cathelicidin-BF (BF-30) Lys16 mutant derived from the snake venom of Bungarus fasciatus. Our previous study found that BF-30 selectively inhibited the proliferation of the metastatic melanoma cell line B16F10 in vitro and in vivo, but had a negligible effect on human lung cells. In the present study, it was demonstrated for the first time that Cbf-K16 selectively inhibits the proliferation of lung carcinoma cells in vitro, with low toxicity to normal cells. The half-maximal inhibitory concentrations (IC50) of Cbf-K16 against H460 human non-small cell lung carcinoma cells and mouse Lewis lung cancer cells were only 16.5 and 10.5 µM, respectively, which were much less compared to that of BF-30 (45 and 40.3 µM). Data using a transmission electron microscope (TEM) assay showed that, at 20 and 40 µM, Cbf-K16 induced the rupture of the cytoplasmic membrane, which was consistent with data obtained from lactate dehydrogenase (LDH) release assays. The LDH release increased from 17.8 to 52.9% as the duration and dosage of Cbf-K16 increased. Annexin V-ﬂuorescein and propidium iodide staining assays indicated that there were no obvious apoptotic effects at the different dosages and times tested. In H460 cells, the rate of genomic DNA binding increased from 51.9 to 86.8% as the concentration of Cbf-K16 increased from 5 to 10 µM. These data indicate that Cbf-K16 selectively inhibits the proliferation of lung carcinoma cells via cytoplasmic membrane permeabilization and DNA binding, rather than apoptosis. Although Cbf-K16 displayed significant cytotoxic activity (40 µM) against tumor cells, in splenocytes no significant inhibitory effect was observed and hemolysis was only 5.6%. These results suggest that Cbf-K16 is a low-toxicity anti-lung cancer drug candidate.

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1	Yan JX, Wang KR, Chen R, Song JJ, Zhang BZ, Dang W, Zhang W and Wang R: Membrane active antitumor activity of NK-18, a mammalian NK-lysin-derived cationic antimicrobial peptide. Biochimie. 94:184–191. 2012. View Article : Google Scholar : PubMed/NCBI
2	Lehmann J, Retz M, Sidhu SS, Suttmann H, Sell M, Paulsen F, Harder J, Unteregger G and Stöckle M: Antitumor activity of the antimicrobial peptide magainin II against bladder cancer cell lines. Eur Urol. 50:141–147. 2006. View Article : Google Scholar : PubMed/NCBI
3	Hoskin DW and Ramamoorthy A: Studies on anticancer activities of antimicrobial peptides. Biochim Biophysica Acta. 1778:357–375. 2008. View Article : Google Scholar : PubMed/NCBI
4	Vanajothi R, Sudha A, Manikandan R, Rameshthangam P and Srinivasan P: Luffa acutangula and lippia nodiflora leaf extract induces growth inhibitory effect through induction of apoptosis on human lung cancer cell line. Biomed Prev Nutr. 2:287–293. 2012. View Article : Google Scholar
5	Quadrelli S, Lyons G, Colt H, Chimondeguy D and Silva C: Lung cancer as a second primary malignancy: increasing prevalence and its influence on survival. Ann Surg Oncol. 16:1033–1038. 2009. View Article : Google Scholar : PubMed/NCBI
6	Kim HM, Lim J, Park SK, Kang JS, Lee K, Lee CW, Lee KH, Yun MJ, Yang KH, Han G, Kwon SW, Kim Y and Han SB: Antitumor activity of cytokine-induced killer cells against human lung cancer. Int Immunopharmacol. 7:1802–1807. 2007. View Article : Google Scholar : PubMed/NCBI
7	Doroshow JH, Juhasz A, Ge Y, Holbeck S, Lu J, Antony S, Wu Y, Jiang G and Roy K: Antiproliferative mechanisms of action of the flavin dehydrogenase inhibitors diphenylene iodonium and di-2-thienyliodonium based on molecular profiling of the NCI-60 human tumor cell panel. Biochem Pharmacol. 83:1195–1207. 2012. View Article : Google Scholar
8	Maione P, Rossi A, Airoma G, Ferrara C, Castaldo V and Gridelli C: The role of targeted therapy in non-small cell lung cancer. Crit Rev Oncol Hematol. 51:29–44. 2004. View Article : Google Scholar : PubMed/NCBI
9	Koh PK, Faivre-Finn C, Blackhall FH and Ruysscher D: Targeted agents in non-small cell lung cancer (NSCLC): clinical developments and rationale for the combination with thoracic radiotherapy. Cancer Treat Rev. 38:626–640. 2012. View Article : Google Scholar : PubMed/NCBI
10	Zhang W, Shi Y, Chen Y, Yu S, Hao J, Luo J, Sha X and Fang X: Enhanced antitumor efficacy by paclitaxel-loaded pluronic P123/F127 mixed micelles against non-small cell lung cancer based on passive tumor targeting and modulation of drug resistance. Eur J Pharm Biopharm. 75:341–353. 2010. View Article : Google Scholar
11	Dempke WC, Suto T and Reck M: Targeted therapies for non-small cell lung cancer. Lung Cancer. 67:257–274. 2010. View Article : Google Scholar : PubMed/NCBI
12	Wang K, Yan J, Liu X, Zhang J, Chen R, Zhang B, Dang W, Zhang W, Kai M, Song J and Wang R: Novel cytotoxity exhibition mode of polybia-CP, a novel antimicrobial peptide from the venom of the social wasp Polybia paulista. Toxicology. 288:27–33. 2011. View Article : Google Scholar : PubMed/NCBI
13	Zasloff M: Antimicrobial peptides of multicellular organisms. Nature. 415:389–395. 2002. View Article : Google Scholar : PubMed/NCBI
14	Chen C, Hu J, Zhang S, Zhou P, Zhao X, Xu H, Zhao X, Yaseen M and Lu JR: Molecular mechanisms of antibacterial and antitumor actions of designed surfactant-like peptides. Biomaterials. 33:592–603. 2012. View Article : Google Scholar : PubMed/NCBI
15	Zhou H, Dou J, Wang J, Chen L, Wang H, Zhou W, Li Y and Zhou C: The antibacterial activity of BF-30 in vitro and in infected burned rats is through interference with cytoplasmic membrane integrity. Peptides. 32:1131–1138. 2011. View Article : Google Scholar : PubMed/NCBI
16	Almaaytah A, Zhou M, Wang L, Chen T, Walker B and Shaw C: Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: biochemical and functional characterization of natural peptides and a single site-substituted analog. Peptides. 35:291–299. 2012.PubMed/NCBI
17	Okumura K: Cathelicidins - Therapeutic antimicrobial and antitumor host defense peptides for oral diseases. Jpn Dental Sci Rev. 47:67–81. 2011. View Article : Google Scholar
18	Chan DI, Prenner EJ and Vogel HJ: Tryptophan- and arginine-rich antimicrobial peptides: structures and mechanisms of action. Biochim Biophys Acta. 1758:1184–1202. 2006. View Article : Google Scholar : PubMed/NCBI
19	Wang Y, Hong J, Liu X, Yang H, Liu R, Wu J, Wang A, Lin D and Lai R: Snake cathelicidin from Bungarus fasciatus is a potent peptide antibiotics. PLoS One. 3:e32172008.PubMed/NCBI
20	Wang Y, Zhang Z, Chen L, Guang H, Li Z, Yang H, Li J, You D, Yu H and Lai R: Cathelicidin-BF, a snake cathelicidin-derived antimicrobial peptide, could be an excellent therapeutic agent for Acne Vulgaris. PLoS One. 6:e221202011. View Article : Google Scholar
21	Hao Q, Wang H, Wang J, Dou J, Zhang M, Zhou W and Zhou C: Effective antimicrobial activity of Cbf-K₁₆ and Cbf-A₇A₁₃ against NDM-1-carrying Escherichia coli by DNA binding after penetrating the cytoplasmic membrane in vitro. J Pept Sci. 19:173–180. 2013.PubMed/NCBI
22	Wang H, Ke M, Tian Y, Wang J, Li B, Wang Y, Dou J and Zhou C: BF-30 selectively inhibits melanoma cell proliferation via cytoplasmic membrane permeabilization and DNA-binding in vitro and in B16F10-bearing mice. Eur J Pharmacol. 707:1–10. 2013. View Article : Google Scholar : PubMed/NCBI
23	Lin WJ, Chien YL, Pan CY, Lin TL, Chen JY, Chiu SJ and Hui CF: Epinecidin-1, an antimicrobial peptide from fish (Epinephelus coioides) which has an antitumor effect like lytic peptides in human fibrosarcoma cells. Peptides. 30:283–290. 2009.PubMed/NCBI
24	Chen JY, Lin WJ and Lin TL: A fish antimicrobial peptide, tilapia hepcidin TH2-3, shows potent antitumor activity against human fibrosarcoma cells. Peptides. 30:1636–1642. 2009. View Article : Google Scholar : PubMed/NCBI
25	Wang C, Li HB, Li S, Tian LL and Shang DJ: Antitumor effects and cell selectivity of temporin-1CEa, an antimicrobial peptide from the skin secretions of the Chinese brown frog (Rana chensinensis). Biochimie. 94:434–441. 2012. View Article : Google Scholar : PubMed/NCBI
26	Zhang Y, Qu Y, Zhang J and Wang X: Ardipusilloside I purified from Ardisia pusilla competitively binds VEGFR and induces apoptosis in NCI-H460 cells. Phytomedicine. 17:519–526. 2010.
27	Bolt AM, Byrd RM and Klimecki WT: Autophagy is the predominant process induced by arsenite in human lymphoblastoid. Toxicol Appl Pharmacol. 244:366–373. 2010. View Article : Google Scholar : PubMed/NCBI
28	Chang WT, Pan CY, Rajanbabu V, Cheng CW and Chen JY: Tilapia (Oreochromis mossambicus) antimicrobial peptide, hepcidin 1–5, shows antitumor activity in cancer cells. Peptides. 32:342–352. 2011.PubMed/NCBI
29	Hsu JC, Lin LC, Tzen JT and Chen JY: Characteristics of the antitumor activities in tumor cells and modulation of the inflammatory response in RAW264.7 cells of a novel antimicrobial peptide, chrysophsin-1, from the red sea bream (Chrysophrys major). Peptides. 32:900–910. 2011. View Article : Google Scholar : PubMed/NCBI
30	Chen JY, Lin WJ, Wu JL, Her GM and Hui CF: Epinecidin-1 peptide induces apoptosis which enhances antitumor effects in human leukemia U937 cells. Peptides. 30:2365–2373. 2009. View Article : Google Scholar : PubMed/NCBI
31	Paredes-Gamero EJ, Martins MN, Cappabianco FA, Ide JS and Miranda A: Characterization of dual effects induced by antimicrobial peptides: regulated cell death or membrane disruption. Biochim Biophys Acta. 1820:1062–1072. 2012. View Article : Google Scholar : PubMed/NCBI
32	Liu H, Zhou BH, Qiu X, Wang HS, Zhang F, Fang R, Wang XF, Cai SH, Du J and Bu XZ: T63, a new 4-arylidene curcumin analogue, induces cell cycle arrest and apoptosis through activation of the reactive oxygen species-FOXO3a pathway in lung cancer. Free Radic Biol Med. 53:2204–2217. 2012. View Article : Google Scholar : PubMed/NCBI
33	Wang YQ, Su J, Wu F, Lu P, Yuan LF, Yuan WE, Sheng J and Jin T: Biscarbamate cross-linked polyethylenimine derivative with low molecular weight, low cytotoxicity, and high efficiency for gene delivery. Int J Nanomedicine. 7:693–704. 2012.PubMed/NCBI
34	Hsu JC, Lin LC, Tzen JT and Chen JY: Pardaxin-induced apoptosis enhances antitumor activity in HeLa cells. Peptides. 32:1110–1116. 2011. View Article : Google Scholar : PubMed/NCBI
35	Kim YS, Jin HO, Seo SK, Woo SH, Choe TB, An S, Hong SI, Lee SJ, Lee KH and Park IC: Sorafenib induces apoptotic cell death in human non-small cell lung cancer cells by down-regulating mammalian target of rapamycin (mTOR)-dependent survivin expression. Biochem Pharmacol. 82:216–226. 2011. View Article : Google Scholar : PubMed/NCBI
36	Custodio A, Méndez M and Provencio M: Targeted therapies for advanced non-small-cell lung cancer: current status and future implications. Cancer Treat Rev. 38:36–53. 2012. View Article : Google Scholar : PubMed/NCBI
37	de Wilt LH, Jansen G, Assaraf YG, van Meerloo J, Cloos J, Schimmer AD, Chan ET, Kirk CJ, Peters GJ and Kruyt FA: Proteasome-based mechanisms of intrinsic and acquired bortezomib resistance in non-small cell lung cancer. Biochem Pharmacol. 83:207–217. 2012.PubMed/NCBI
38	Leung HW, Yang WH, Lai MY, Lin CJ and Lee HZ: Inhibition of 12-lipoxygenase during baicalein-induced human lung non-small carcinoma H460 cell apoptosis. Food Chem Toxicol. 45:403–411. 2007. View Article : Google Scholar : PubMed/NCBI
39	Silvestri GA and Rivera MP: Targeted therapy for the treatment of advanced non-small cell lung cancer: a review of the epidermal growth factor receptor antagonists. Chest. 128:3975–3984. 2005. View Article : Google Scholar : PubMed/NCBI
40	Chen YL, Li JH, Yu CY, Lin CJ, Chiu PH, Chen PW, Lin CC and Chen WJ: Novel cationic antimicrobial peptide GW-H1 induced caspase-dependent apoptosis of hepatocellular carcinoma cell lines. Peptides. 36:257–265. 2012. View Article : Google Scholar : PubMed/NCBI
41	Schweizer F: Cationic amphiphilic peptides with cancer-selective toxicity. Eur J Pharmacol. 625:190–194. 2009. View Article : Google Scholar : PubMed/NCBI
42	Liu Y, Xia X, Xu L and Wang Y: Design of hybrid β-hairpin peptides with enhanced cell specificity and potent anti-inflammatory activity. Biomaterials. 34:237–250. 2013.