Abstract
Lung cancer has a relatively poor prognosis with a low survival rate and drugs that target other cell death mechanism like autophagy may help improving current therapeutic strategy. This study investigated the anti-proliferative effect of Licarin A (LCA) from Myristica fragrans in non-small cell lung cancer cell lines—A549, NCI-H23, NCI-H520 and NCI-H460. LCA inhibited proliferation of all the four cell lines in a dose and time dependent manner with minimum IC50 of 20.03 ± 3.12, 22.19 ± 1.37 µM in NCI-H23 and A549 cells respectively. Hence NCI-H23 and A549 cells were used to assess the ability LCA to induce autophagy and apoptosis. LCA treatment caused G1 arrest, increase in Beclin 1, LC3II levels and degradation of p62 indicating activation of autophagy in both NCI-H23 and A549 cells. In addition, LCA mediated apoptotic cell death was confirmed by MMP loss, increased ROS, cleaved PARP and decreased pro-caspase3. To understand the role of LCA induced autophagy and its association with apoptosis, cells were analysed following treatment with a late autophagy inhibitor-chloroquine and also after Beclin 1 siRNA transfection. Data indicated that inhibition of autophagy resulted in reduced anti-proliferative as well as pro-apoptotic ability of LCA. These findings confirmed that LCA brought about autophagy dependent apoptosis in non-small cell lung cancer cells and hence it may serve as a potential drug candidate for non-small cell lung cancer therapy.
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15 March 2018
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Abbreviations
- LCA:
-
Licarin A
- CQ:
-
Chloroquine
- LC3:
-
Microtubule-associated protein 1 light chain
- EGFR:
-
Epidermal growth factor receptor
- p62:
-
Sequestosome-1/ubiquitin-binding protein
- p53:
-
Tumor suppressor protein
- ROS:
-
Reactive oxygen species
- DCFH-DA:
-
2′,7′-Dichlorofluorescein-diacetate
- PI:
-
Propidium iodide
- PARP:
-
Poly(ADP-ribose) polymerase
- NMR:
-
Nuclear magnetic resonance
- FTIR:
-
Fourier transform infrared
- HPLC:
-
High performance liquid chromatography
References
García-Campelo R, Bernabé R, Cobo M et al (2015) SEOM clinical guidelines for the treatment of non-small cell lung cancer (NSCLC) 2015. Clin Transl Oncol 17:1020–1029. https://doi.org/10.1007/s12094-015-1455-z
Liu SV, Zhao W, Petrini I et al (2017) Third-generation tyrosine kinase inhibitors targeting epidermal growth factor receptor mutations in non-small cell lung cancer. Front Oncol. https://doi.org/10.3389/fonc.2017.00113
Wang M, Wang G, Ma H, Shan B (2017) Crizotinib versus chemotherapy on ALK-positive NSCLC: a systematic review of efficacy and safety. Curr Cancer Drug Targets. https://doi.org/10.2174/1568009617666170623115846
Singh Malik P, Raina V (2012) Lung cancer: prevalent trends & emerging concepts. Int Agency Res Cancer Indian Counc Med Res 141:2009–2011. https://doi.org/10.4103/0971-5916.154479
Howlader N, Noone AM, Krapcho M, Miller D, Bishop K, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS FEC SEER Cancer Statistics Review, 1975-2014 National Cancer Institute, Bethesda, MD, Based on November 2016 SEER data submission, posted to SEER web site, April 2017.
Housman G, Byler S, Heerboth S et al (2014) Drug resistance in cancer: an overview. Cancers 6:1769–1792. https://doi.org/10.3390/cancers6031769
Indran IR, Tufo G, Pervaiz S, Brenner C (2011) Recent advances in apoptosis, mitochondria and drug resistance in cancer cells. Biochim Biophys Acta 1807:735–745. https://doi.org/10.1016/j.bbabio.2011.03.010
Glick D, Barth S, Macleod KF (2010) Autophagy: cellular and molecular mechanisms. J Pathol 221:3–12. https://doi.org/10.1002/path.2697.Autophagy
Morselli E, Galluzzi L, Kepp O et al (2009) Anti- and pro-tumor functions of autophagy. Biochim Biophys Acta 1793:1524–1532. https://doi.org/10.1016/j.bbamcr.2009.01.006
Ishaq M, Khan MA, Sharma K et al (2014) Gambogic acid induced oxidative stress dependent caspase activation regulates both apoptosis and autophagy by targeting various key molecules (NF-κB, Beclin-1, p62 and NBR1) in human bladder cancer cells. Biochim Biophys Acta 1840:3374–3384. https://doi.org/10.1016/j.bbagen.2014.08.019
Peng X, Gong F, Chen Y et al (2014) Autophagy promotes paclitaxel resistance of cervical cancer cells: involvement of Warburg effect activated hypoxia-induced factor 1-α-mediated signaling. Cell Death Dis 5:e1367. https://doi.org/10.1038/cddis.2014.297
Sharma N, Thomas S, Golden EB et al (2012) Inhibition of autophagy and induction of breast cancer cell death by mefloquine, an antimalarial agent. Cancer Lett 326:143–154. https://doi.org/10.1016/j.canlet.2012.07.029
Li LH, Wu P, Lee JY et al (2014) Hinokitiol induces DNA damage and autophagy followed by cell cycle arrest and senescence in gefitinib-resistant lung adenocarcinoma cells. PLoS ONE. https://doi.org/10.1371/journal.pone.0104203
Wang FZ, Fei HR, Cui YJ et al (2014) The checkpoint 1 kinase inhibitor LY2603618 induces cell cycle arrest, DNA damage response and autophagy in cancer cells. Apoptosis 19:1389–1398. https://doi.org/10.1007/s10495-014-1010-3
Jinous Asgarpanah (2012) Phytochemistry and pharmacologic properties of Myristica fragrans Hoyutt.: a review. Afr J Biotechnol 11:12787–12793. https://doi.org/10.5897/AJB12.1043
Ma CJ, Sung SH, Kim YC (2004) Neuroprotective lignans from the bark of Machilus thunbergii. Planta Med 70:79–80. https://doi.org/10.1055/s-2004-815463
Néris PLN, Caldas JPA, Rodrigues YKS et al (2013) Neolignan Licarin A presents effect against Leishmania (Leishmania) major associated with immunomodulation in vitro. Exp Parasitol 135:307–313. https://doi.org/10.1016/j.exppara.2013.07.007
Sawasdee K, Chaowasku T, Lipipun V et al (2013) New neolignans and a lignan from Miliusa fragrans, and their anti-herpetic and cytotoxic activities. Tetrahedron Lett 54:4259–4263. https://doi.org/10.1016/j.tetlet.2013.05.144
León-Díaz R, Meckes-Fischer M, Valdovinos-Martínez L et al (2013) Antitubercular activity and the subacute toxicity of (-)-licarin a in balb/c mice: a neolignan isolated from Aristolochia taliscana. Arch Med Res 44:99–104. https://doi.org/10.1016/j.arcmed.2012.12.006
Luo L, Li Y, Liu Z-Q et al (2017) Honokiol induces apoptosis, G1 arrest, and autophagy in KRAS mutant lung cancer cells. Front Pharmacol 8:199. https://doi.org/10.3389/fphar.2017.00199
Zhou J, Hu H, Long J et al (2013) Vitexin 6, a novel lignan, induces autophagy and apoptosis by activating the Jun N-terminal kinase pathway. Anticancer Drugs 24:928–936. https://doi.org/10.1097/CAD.0b013e328364e8d3
Nguyen PH, Kang HW, Le TVT et al (2011) Simple process for the decrease of myristicin content from Myristica fragrans (nutmeg) and its activity with amp-activated protein kinase (AMPK). J Food Biochem 35:1715–1722. https://doi.org/10.1111/j.1745-4514.2010.00496.x
Mah LY, Ryan KM (2012) Autophagy and cancer. Cold Spring Harb Perspect Biol 4:a008821. https://doi.org/10.1101/cshperspect.a008821
Brech A, Ahlquist T, Lothe RA, Stenmark H (2009) Autophagy in tumour suppression and promotion. Mol Oncol 3:366–375. https://doi.org/10.1016/j.molonc.2009.05.007
Harhaji-Trajkovic L, Arsikin K, Kravic-Stevovic T et al (2012) Chloroquine-mediated lysosomal dysfunction enhances the anticancer effect of nutrient deprivation. Pharm Res 29:2249–2263. https://doi.org/10.1007/s11095-012-0753-1
Giono LE, Manfredi JJ (2006) The p53 tumor suppressor participates in multiple cell cycle checkpoints. J Cell Physiol 209:13–20. https://doi.org/10.1002/jcp.20689
Kongara S, Karantza V (2012) The interplay between autophagy and ROS in tumorigenesis. Front Oncol 2:171. https://doi.org/10.3389/fonc.2012.00171
Poillet-Perez L, Despouy G, Delage-Mourroux R, Boyer-Guittaut M (2015) Interplay between ROS and autophagy in cancer cells, from tumor initiation to cancer therapy. Redox Biol 4:184–192. https://doi.org/10.1016/j.redox.2014.12.003
Eruslanov E, Kusmartsev S (2010) Identification of ROS using oxidized DCFDA and flow-cytometry. Methods Mol Biol 594:57–72. https://doi.org/10.1007/978-1-60761-411-1_4
Kasibhatla S (2006) Acridine orange/ethidium bromide (AO/EB) staining to detect apoptosis. Cold Spring Harb Protoc 2006:pdb.prot4493–prot4493. https://doi.org/10.1101/pdb.prot4493
Chazotte B (2011) Labeling nuclear DNA using DAPI. Cold Spring Harb Protoc 6:pdb.prot5556. https://doi.org/10.1101/pdb.prot5556
Baracca A, Sgarbi G, Solaini G, Lenaz G (2003) Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F0 during ATP synthesis. Biochim Biophys Acta 1606:137–146. https://doi.org/10.1016/S0005-2728(03)00110-5
Yang ZJ, Chee CE, Huang S, Sinicrope FA (2011) The role of autophagy in cancer: therapeutic implications. Mol Cancer Ther 10:1533–1541. https://doi.org/10.1158/1535-7163.MCT-11-0047
Guo XL, Li D, Sun K et al (2013) Inhibition of autophagy enhances anticancer effects of bevacizumab in hepatocarcinoma. J Mol Med 91:473–483. https://doi.org/10.1007/s00109-012-0966-0
Xiao D, Bommareddy A, Kim SH et al (2012) Benzyl isothiocyanate causes FoxO1-mediated autophagic death in human breast cancer cells. PLoS ONE 7:e32597. https://doi.org/10.1371/journal.pone.0032597
Wang Q, Chen Z, Diao X, Huang S (2011) Induction of autophagy-dependent apoptosis by the survivin suppressant YM155 in prostate cancer cells. Cancer Lett 302:29–36. https://doi.org/10.1016/j.canlet.2010.12.007
Kastan MB, Bartek J (2004) Cell-cycle checkpoints and cancer. Nature 432:316–323. https://doi.org/10.1038/nature03097
Vermeulen K, Berneman ZN, Van Bockstaele DR (2003) Cell cycle and apoptosis. Cell Prolif 36:165–175. https://doi.org/10.1046/j.1365-2184.2003.00267.x
Haupt S, Berger M, Goldberg Z, Haupt Y (2003) Apoptosis—the p53 network. J Cell Sci 116:4077–4085. https://doi.org/10.1242/jcs.00739
Chun J, Kang M, Kim YS (2014) A triterpenoid saponin from Adenophora triphylla var. japonica suppresses the growth of human gastric cancer cells via regulation of apoptosis and autophagy. Tumor Biol 35:12021–12030. https://doi.org/10.1007/s13277-014-2501-0
Li C, Chen J, Lu B et al (2014) Molecular switch role of Akt in Polygonatum odoratum lectin-induced apoptosis and autophagy in human non-small cell lung cancer A549 cells. PLoS ONE 9:1–12. https://doi.org/10.1371/journal.pone.0101526
Liu B, Cheng Y, Zhang B et al (2009) Polygonatum cyrtonema lectin induces apoptosis and autophagy in human melanoma A375 cells through a mitochondria-mediated ROS-p38-p53 pathway. Cancer Lett 275:54–60. https://doi.org/10.1016/j.canlet.2008.09.042
Acknowledgements
The research work was funded by Department of Biotechnology, Govt. of India, and DBT-IPLS program (BT/PR14554/INF/22/125/2010). We thank Department of Biochemistry and Molecular Biology for fluorescence microscope facility and Central Instrumentation Facility (CIF), Pondicherry University. We also thank Translational Research Platform for veterinary Biologicals-TANUVAS, Chennai for confocal microscopy facility.
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A correction to this article is available online at https://doi.org/10.1007/s10495-018-1452-0.
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Maheswari, U., Ghosh, K. & Sadras, S.R. Licarin A induces cell death by activation of autophagy and apoptosis in non-small cell lung cancer cells. Apoptosis 23, 210–225 (2018). https://doi.org/10.1007/s10495-018-1449-8
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DOI: https://doi.org/10.1007/s10495-018-1449-8