Abstract
Flavokawain B (FKB), a naturally occurring chalcone in kava extracts, has been reported to possess anticancer activity. However, the effect of FKB on gastric cancer remains unclear. We examined the in vitro and in vivo anticancer activity and autophagy involvement of FKB and determined the underlying molecular mechanisms. FKB is potently cytotoxic to human gastric cancer cells (AGS/NCI-N87/KATO-III/TSGH9201) and mildly toxic towards normal (Hs738) cells and primary mouse hepatocytes. FKB-induced AGS cell death was characterized by autophagy, not apoptosis, as evidenced by increased LC3-II accumulation, GFP-LC3 puncta and acidic vesicular organelles (AVOs) formation, without resulting procaspase-3/PARP cleavage. FKB further caused p62/SQSTM1 activation, mTOR downregulation, ATG4B inhibition, and Beclin-1/Bcl-2 dysregulation. Silencing autophagy inhibitors CQ/3-MA and LC3 (shRNA) significantly reversed the FKB-induced cell death of AGS cells. FKB-triggered ROS generation and ROS inhibition by NAC pre-treatment diminished FKB-induced cell death, LC3 conversion, AVO formation, p62/SQSTM1 activation, ATG4B inhibition and Beclin-1/Bcl-2 dysregulation, which indicated ROS-mediated autophagy in AGS cells. Furthermore, FKB induces G2/M arrest and alters cell-cycle proteins through ROS-JNK signaling. Interestingly, FKB-induced autophagy is associated with the suppression of HER-2 and PI3K/AKT/mTOR signaling cascades. FKB inhibits apoptotic Bax expression, and Bax-transfected AGS cells exhibit both apoptosis and autophagy; thus, FKB-inactivated Bax results in apoptosis inhibition. In vivo data demonstrated that FKB effectively inhibited tumor growth, prolonged the survival rate, and induced autophagy in AGS-xenografted mice. Notably, silencing of LC3 attenuated FKB-induced autophagy in AGS-xenografted tumors. FKB may be a potential chemopreventive agent in the activation of ROS-mediated autophagy of gastric cancer cells.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- FKB:
-
Flavokawain B
- MTT:
-
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- LC3:
-
Microtubule-associated light chain 3
- AVO:
-
Acidic vesicular organelle
- PARP:
-
Poly (ADP-ribose) polymerase
- CQ:
-
Chloroquine
- 3-MA:
-
3-Methyladenine
- ROS:
-
Reactive oxygen species
- DCFH2-DA:
-
2′,7′-dihydrofluorescein-diacetate
- NAC:
-
N-acetyl-l-cysteine
- p38 MAPK:
-
p38 mitogen-activated protein kinase
- JNK:
-
c-jun N-terminal kinase
- ERK:
-
Extracellular signal-regulated protein kinase
- CDC25C:
-
Cell division cycle 25 °C
- PI3K:
-
Phosphatidylinositol 3-kinase
- HER2:
-
Human epidermal growth factor receptor 2
References
Abu N, Ho WY, Yeap SK, Akhtar MN, Abdullah MP, Omar AR, Alitheen NB (2013) The flavokawains: uprising medicinal chalcones. Cancer cell Int 13:102 doi:10.1186/1475-2867-13-102
Abu N et al (2015) In vivo antitumor and antimetastatic effects of flavokawain B in 4T1 breast cancer cell-challenged mice. Drug Des Devel Ther 9:1401–1417 doi:10.2147/DDDT.S67976
Carreñ OI LE, Martelloni A, Salas B, Simõ es BG, Vergona PR (2014) German court repeals the withdrawal of marketing authorisation of kava-containing medicinal products Trade. Perspectives 13:2–5
Choi KS (2012) Autophagy and cancer. Exp Mol Med 44:109–120 doi:10.3858/emm.2012.44.2.033
Clayton NP, Yoshizawa K, Kissling GE, Burka LT, Chan PC, Nyska A (2007) Immunohistochemical analysis of expressions of hepatic cytochrome P450 in F344 rats following oral treatment with kava extract. Exp Toxicol Pathol 58:223–236 doi:10.1016/j.etp.2006.08.002
DiSilvestro RA, Zhang W, DiSilvestro DJ (2007) Kava feeding in rats does not cause liver injury nor enhance galactosamine-induced hepatitis. Food Chem Toxicol 45:1293–1300. doi:10.1016/j.fct.2007.01.015
Green DR (2000) Apoptotic pathways: paper wraps stone blunts scissors. Cell 102:1–4. doi:10.1016/S0092-8674(00)00003-9
Hale AN, Ledbetter DJ, Gawriluk TR, Rucker EB 3rd (2013) Autophagy: regulation and role in development. Autophagy 9:951–972. doi:10.4161/auto.24273
Hasima N, Ozpolat B (2014) Regulation of autophagy by polyphenolic compounds as a potential therapeutic strategy for cancer. Cell Death Dis 5:e1509 doi:10.1038/cddis.2014.467
Hseu YC et al (2012) The chalcone flavokawain B induces G2/M cell-cycle arrest and apoptosis in human oral carcinoma HSC-3 cells through the intracellular ROS generation and downregulation of the Akt/p38 MAPK signaling pathway. J Agric Food Chem 60:2385–2397. doi:10.1021/jf205053r
Hsin IL et al (2010) N-acetyl cysteine mitigates curcumin-mediated telomerase inhibition through rescuing of Sp1 reduction in A549 cells. Mutat Res. doi:10.1016/j.mrfmmm.2010.03.011
Huang CC, Lien HH, Sung YC, Liu HT, Chie WC (2007) Quality of life of patients with gastric cancer in Taiwan: validation and clinical application of the Taiwan Chinese version of the EORTC QLQ-C30 and EORTC QLQ-STO22. Psychooncology 16:945–949. doi:10.1002/pon.1158
Jain K, Paranandi KS, Sridharan S, Basu A (2013) Autophagy in breast cancer and its implications for therapy. A J Cancer Res 3:251–265
Kang R, Zeh H, Lotze M, Tang D (2011) The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ 18:571–580
Karna P, Zughaier S, Pannu V, Simmons R, Narayan S, Aneja R (2010) Induction of reactive oxygen species-mediated autophagy by a novel microtubule-modulating agent. J Biol Chem 285:18737–18748. doi:10.1074/jbc.M109.091694
Klionsky DJ, Emr SD (2000) Autophagy as a regulated pathway of cellular degradation. Science 290:1717–1721
Komoto M et al (2009) HER2 overexpression correlates with survival after curative resection of pancreatic cancer. Cancer Sci 100:1243–1247. doi:10.1111/j.1349-7006.2009.01176.x
Kondo Y, Kanzawa T, Sawaya R, Kondo S (2005) The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 5:726–734. doi:10.1038/nrc1692
Lin E et al. (2012) Flavokawain B inhibits growth of human squamous carcinoma cells: involvement of apoptosis and cell cycle dysregulation in vitro and in vivo. J Nutr Biochem 23:368–378 doi:10.1016/j.jnutbio.2011.01.002
Marzo I et al (1998) Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281:2027–2031
Mingo-Sion AM, Marietta PM, Koller E, Wolf DM, Van Den Berg CL (2004) Inhibition of JNK reduces G2/M transit independent of p53, leading to endoreduplication, decreased proliferation, and apoptosis in breast cancer cells. Oncogene 23:596–604. doi:10.1038/sj.onc.1207147
Mohan N, Chakrabarti M, Banik NL, Ray SK (2013) Combination of LC3 shRNA plasmid transfection and genistein treatment inhibited autophagy and increased apoptosis in malignant neuroblastoma in cell culture and animal models. PLoS One 8:e78958. doi:10.1371/journal.pone.0078958
Munshi A, Ramesh R (2013) Mitogen-activated protein kinases and their role in radiation response. Genes Cancer 4:401–408 doi:10.1177/1947601913485414
Narayanapillai SC, Leitzman P, O’Sullivan MG, Xing C (2014) Flavokawains a and B in kava, not dihydromethysticin, potentiate acetaminophen-induced hepatotoxicity in C57BL/6 mice. Chem Res Toxicol 27:1871–1876 doi:10.1021/tx5003194
Nowakowska Z (2007) A review of anti-infective and anti-inflammatory chalcones. Eur J Med Chem 42:125–137 doi:10.1016/j.ejmech.2006.09.019
Ohsumi Y (2001) Molecular dissection of autophagy: two ubiquitin-like systems. Nat Rev Mol Cell Biol 2:211–216. doi:10.1038/35056522
Oltvai ZN, Milliman CL, Korsmeyer SJ (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609–619
Ozpolat B, Dalby K, Berestein GL (2012) Induction of autophagy by polyphenolic compounds in cancer: a novel Strategy to induce cell death and to treat Cancer. In: Diederich M, Noworyta K (eds) Natural compounds as inducers of cell death, vol 1. Springer, Dordrecht, pp 237–261
Pan MH, Ho CT (2008) Chemopreventive effects of natural dietary compounds on cancer development. Chem Soc Rev 37:2558–2574 doi:10.1039/b801558a
Pattingre S et al (2005) Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 122:927–939. doi:10.1016/j.cell.2005.07.002
Pelicano H, Carney D, Huang P (2004) ROS stress in cancer cells and therapeutic implications. Drug Resist Updat 7:97–110 doi:10.1016/j.drup.2004.01.004
Qiao S et al (2015) A REDD1/TXNIP pro-oxidant complex regulates ATG4B activity to control stress-induced autophagy and sustain exercise capacity. Nat Commun 6:7014. doi:10.1038/ncomms8014
Rothe K et al (2014) The core autophagy protein ATG4B is a potential biomarker and therapeutic target in CML stem/progenitor cells. Blood 123:3622–3634. doi:10.1182/blood-2013-07-516807
Singh YN, Devkota AK (2003) Aqueous kava extracts do not affect liver function tests in rats. Planta Med 69:496–499 doi:10.1055/s-2003-40658
Sorrentino L, Capasso A, Schmidt M (2006) Safety of ethanolic kava extract: results of a study of chronic toxicity in rats. Phytomedicine 13:542–549 doi:10.1016/j.phymed.2006.01.006
Steiner GG (2000) The correlation between cancer incidence and kava consumption. Hawaii Med J 59:420–422
Sukawa Y et al (2014) HER2 expression and PI3K-Akt pathway alterations in gastric cancer. Digestion 89:12–17. doi:10.1159/000356201
Tanida I, Ueno T, Kominami E (2004) LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol 36:2503–2518 doi:10.1016/j.biocel.2004.05.009
Teschke R (2010) Kava hepatotoxicity–a clinical review. Ann Hepatol 9:251–265
Thiyagarajan V, Tsai MJ, Weng CF (2015) Antroquinonol targets FAK-signaling pathway suppressed cell migration, invasion, and tumor growth of C6 glioma. PLoS One 10:e0141285. doi:10.1371/journal.pone.0141285.PONE-D-15-20248
Thiyagarajan V, Sivalingam KS, Viswanadha VP, Weng CF (2016) 16-hydroxy-cleroda-3,13-dien-16,15-olide induced glioma cell autophagy via ROS generation and activation of p38 MAPK and ERK-1/2. Environ Toxicol Pharmacol 45:202–211 doi:10.1016/j.etap.2016.06.005
Wagner EF, Nebreda AR (2009) Signal integration by JNK and p38 MAPK pathways in cancer development. Nat Rev Cancer 9:537–549. doi:10.1038/nrc2694
Xie CM, Chan WY, Yu S, Zhao J, Cheng CH (2011) Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation. Free Radic Biol Med 51:1365–1375. doi:10.1016/j.freeradbiomed.2011.06.016
Zhang X, Chen LX, Ouyang L, Cheng Y, Liu B (2012) Plant natural compounds: targeting pathways of autophagy as anti-cancer therapeutic agents. Cell Prolif 45:466–476 doi:10.1111/j.1365-2184.2012.00833.x
Zhang Z et al (2013) Wentilactone B induces G2/M phase arrest and apoptosis via the Ras/Raf/MAPK signaling pathway in human hepatoma SMMC-7721 cells. Cell Death Dis 4:e657 doi:10.1038/cddis.2013.182
Zhang H et al (2014) Harmine induces apoptosis and inhibits tumor cell proliferation, migration and invasion through down-regulation of cyclooxygenase-2 expression in gastric cancer. Phytomedicine 21:348–355 doi:10.1016/j.phymed.2013.09.007
Zhao Y, Wu Z, Zhang Y, Zhu L (2013) HY-1 induces G(2)/M cell cycle arrest in human colon cancer cells through the ATR-Chk1-Cdc25C and Weel pathways. Cancer Sci 104:1062–1066. doi:10.1111/cas.12182
Zhou P et al (2010) Flavokawain B, the hepatotoxic constituent from kava root, induces GSH-sensitive oxidative stress through modulation of IKK/NF-kappaB and MAPK signaling pathways. FASEB J 24:4722–4732. doi:10.1096/fj.10-163311
Zhou H et al (2014) Ciclopirox induces autophagy through reactive oxygen species-mediated activation of JNK signaling pathway. Oncotarget 5:10140–10150. doi:10.18632/oncotarget.2471
Zi X, Simoneau AR (2005) Flavokawain A, a novel chalcone from kava extract, induces apoptosis in bladder cancer cells by involvement of Bax protein-dependent and mitochondria-dependent apoptotic pathway and suppresses tumor growth in mice. Cancer Res 65:3479–3486. doi:10.1158/0008-5472.CAN-04-3803
Acknowledgements
This work was supported by Grants MOST-104-2320-B-039-040-MY3, MOST-103-2320-B-039-038 -MY3, NSC-103-2622-B-039-001-CC2, CMU103-ASIA-12, and CMU 103-ASIA-09 from the Ministry of Science and Technology, Asia University, and China Medical University, Taiwan to Dr. Hsin-Ling Yang and Dr. You-Cheng Hseu. This study was supported by China Medical University under the Aim for Top University Plan of the Ministry of Education, Taiwan (CHM106-5-3).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that there is no conflict of interest.
Rights and permissions
About this article
Cite this article
Chang, CT., Hseu, YC., Thiyagarajan, V. et al. Chalcone flavokawain B induces autophagic-cell death via reactive oxygen species-mediated signaling pathways in human gastric carcinoma and suppresses tumor growth in nude mice. Arch Toxicol 91, 3341–3364 (2017). https://doi.org/10.1007/s00204-017-1967-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00204-017-1967-0