Abstract
Background
The mammalian target of rapamycin (mTOR) integrates energy level to modulate cell proliferation and autophagy. Cardamonin exhibits anti-proliferative activity through inhibiting mTOR. In this study, the effect of cardamonin on autophagy and its mechanism on mTOR inhibition were investigated.
Methods
Cell viability and proliferation were measured by MTT assay and BrdU incorporation, respectively. Cell apoptosis was assayed by flow cytometry and cell autophagy was detected by electron microscopy and GFP-LC3 fluorescence. The mechanism of cardamonin on mTORC1 inhibition was investigated by Raptor siRNA and Raptor over-expression.
Results
The cell viability and proliferation were inhibited by cardamonin. The autophagosomes and the protein level of LC3-II were increased by cardamonin. Cell apoptosis and the levels of cleaved PARP and Caspase-3 were increased by cardamonin. Cardamonin inhibited the phosphorylation of mTOR and ribosome S6 protein kinase 1 (S6K1) as well as the protein level of regulatory associated protein of mTOR (Raptor). However, cardamonin had no effect on the component of mTORC2 and its downstream substrate Akt. The inhibitory effect of cardamonin on the phosphorylation of mTOR and S6K1 was eliminated by Raptor knockdown with siRNA, whereas this effect of cardamonin was stronger than that of rapamycin and AZD8055 in Raptor over-expression cells. Cell viability was inhibited by cardamonin in both Raptor knockdown and Raptor over-expression cells, which was consistent with the inhibitory effect of cardamonin on mTOR.
Conclusion
These findings demonstrated that the autophagy induced by cardamonin was associated with mTORC1 inhibition through decreasing the protein level of Raptor in SKOV3 cells.
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Abbreviations
- FKBP12:
-
FK506 binding protein 12 kDa
- FRB domain:
-
FKBP12-rapamycin binding domain
- mTOR:
-
mammalian target of rapamycin
- mTORC1/2:
-
mTOR complex 1/2
- NF-κB:
-
nuclear factor-κB
- Raptor:
-
regulatory associated protein of mTOR
- Rictor:
-
rapamycin-insensitive companion of mTOR
- S6K1:
-
ribosome S6 protein kinase 1
References
Levy J.M.M., Towers CG, Thorburn A. Targeting autophagy in cancer. Nat Rev Cancer 2017;17(9):528–42.
Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene 2004;23(16):2891–906.
Fulda S. Autophagy in cancer therapy. Front Oncol 2017;7:128.
Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease. Cell 2017;169(2):361–71.
Prieto-Dominguez N, Ordonez R, Fernandez A, Garcia-Palomo A, Muntane J, Gonzalez-Gallego J, et al. Modulation of autophagy by sorafenib: effects on treatment response. Front Pharmacol 2016;7:151.
Crazzolara R, Bradstock KF, Bendall LJ. RAD001 (Everolimus) induces autophagy in acute lymphoblastic leukemia. Autophagy 2009;5(5):727–8.
Harder LM, Bunkenborg J, Andersen JS. Inducing autophagy: a comparative phosphoproteomic study of the cellular response to ammonia and rapamycin. Autophagy 2014;10(2):339–55.
Bahrami F, Pourgholami MH, Mekkawy AH, Rufener L, Morris DL. Monepantel induces autophagy in human ovarian cancer cells through disruption of the mTOR/p70S6 K signalling pathway. Am J Cancer Res 2014;4(5):558–71.
Frey JW, Jacobs BL, Goodman CA, Hornberger TA. A role for Raptor phosphorylation in the mechanical activation of mTOR signaling. Cell Signal 2014;26(2):313–22.
Dibble CC, Asara JM, Manning BD. Characterization of Rictor phosphorylation sites reveals direct regulation of mTOR complex 2 by S6K1. Mol Cell Biol 2009;29(21):5657–70.
Feng N, Luo J, Guo X. Silybin suppresses cell proliferation and induces apoptosis of multiple myeloma cells via the PI3 K/Akt/mTOR signaling pathway. Mol Med Rep 2016;13(4):3243–8.
Lee da H, Lee CS. Flavonoid myricetin inhibits TNF-alpha-stimulated production of inflammatory mediators by suppressing the Akt, mTOR and NF-kappaB pathways in human keratinocytes. Eur J Pharmacol 2016;784:164–72.
Goncalves LM, Valente IM, Rodrigues JA. An overview on cardamonin. J Med Food 2014;17(6):633–40.
Li YY, Huang SS, Lee MM, Deng JS, Huang GJ. Anti-inflammatory activities of cardamonin from Alpinia katsumadai through heme oxygenase-1 induction and inhibition of NF-kappaB and MAPK signaling pathway in the carrageenan-induced paw edema. Int Immunopharmacol 2015;25(2):332–9.
Niu PG, Zhang YX, Shi DH, Liu Y, Chen YY, Deng J. Cardamonin inhibits metastasis of lewis lung carcinoma cells by decreasing mTOR activity. PLoS One 2015;10(5):e0127778.
Tang Y, Fang Q, Shi D, Niu P, Chen Y, Deng J. mTOR inhibition of cardamonin on antiproliferation of A549 cells is involved in a FKBP12 independent fashion. Life Sci 2014;99(1–2):44–51.
Kim YJ, Kang KS, Choi KC, Ko H. Cardamonin induces autophagy and an antiproliferative effect through JNK activation in human colorectal carcinoma HCT116 cells. Bioorg Med Chem Lett 2015;25(12):2559–64.
Mabuchi S, Hisamatsu T, Kawase C, Hayashi M, Sawada K, Mimura K, et al. The activity of trabectedin as a single agent or in combination with everolimus for clear cell carcinoma of the ovary. Clin Cancer Res 2011;17(13):4462–73.
Mabuchi S, Kawase C, Altomare DA, Morishige K, Sawada K, Hayashi M, et al. mTOR is a promising therapeutic target both in cisplatin-sensitive and cisplatin-resistant clear cell carcinoma of the ovary. Clin Cancer Res 2009;15(17):5404–13.
Chude CI, Amaravadi RK. Targeting autophagy in cancer: update on clinical trials and novel inhibitors. Int J Mol Sci 2017;18(6):1279.
Xu DW, Zhang GQ, Wang ZW, Xu XY, Liu TX. Autophagy in tumorigenesis and cancer treatment. Asian Pac J Cancer Prev 2015;16(6):2167–75.
Lou M, Zhang LN, Ji PG, Feng FQ, Liu JH, Yang C, et al. Quercetin nanoparticles induced autophagy and apoptosis through AKT/ERK/Caspase-3 signaling pathway in human neuroglioma cells: in vitro and in vivo. Biomed Pharmacother 2016;84:1–9.
Zhang XJ, Jia SS. Fisetin inhibits laryngeal carcinoma through regulation of AKT/NF-kappaB/mTOR and ERK1/2 signaling pathways. Biomed Pharmacother 2016;83:1164–74.
Zhang S, Guo C, Chen Z, Zhang P, Li J, Li Y. Vitexin alleviates ox-LDL-mediated endothelial injury by inducing autophagy via AMPK signaling activation. Mol Immunol 2017;85:214–21.
Ko H, Kim YJ, Amor EC, Lee JW, Kim HC, Kim HJ, et al. Induction of autophagy by dimethyl cardamonin is associated with proliferative arrest in human colorectal carcinoma HCT116 and LOVO cells. J Cell Biochem 2011;112(9):2471–9.
Xue ZG, Niu PG, Shi DH, Liu Y, Deng J, Chen YY. Cardamonin inhibits angiogenesis by mTOR downregulation in SKOV3Cells. Planta Med 2016;82(1–2):70–5.
El-Khattouti A, Selimovic D, Haikel Y, Hassan M. Crosstalk between apoptosis and autophagy: molecular mechanisms and therapeutic strategies in cancer. J Cell Death 2013;6:37–55.
Kumar S, Guru SK, Pathania AS, Manda S, Kumar A, Bharate SB, et al. Fascaplysin induces caspase mediated crosstalk between apoptosis and autophagy through the inhibition of PI3 K/AKT/mTOR signaling cascade in human leukemia HL-60 cells. J Cell Biochem 2015;116(6):985–97.
Kumar D, Shankar S, Srivastava RK. Rottlerin induces autophagy and apoptosis in prostate cancer stem cells via PI3 K/Akt/mTOR signaling pathway. Cancer Lett 2014;343(2):179–89.
Zhang DM, Liu JS, Deng LJ, Chen MF, Yiu A, Cao HH, et al. Arenobufagin, a natural bufadienolide from toad venom, induces apoptosis and autophagy in human hepatocellular carcinoma cells through inhibition of PI3 K/Akt/mTOR pathway. Carcinogenesis 2013;34(6):1331–42.
Qin Y, Sun CY, Lu FR, Shu XR, Yang D, Chen L, et al. Cardamonin exerts potent activity against multiple myeloma through blockade of NF-kappaB pathway in vitro. Leuk Res 2012;36(4):514–20.
Li Y, Qin Y, Yang C, Zhang H, Li Y, Wu B, et al. Cardamonin induces ROS-mediated G2/M phase arrest and apoptosis through inhibition of NF-kappaB pathway in nasopharyngeal carcinoma. Cell Death Dis 2017;8(8):e3024.
Shrivastava S, Jeengar MK, Thummuri D, Koval A, Katanaev VL, Marepally S, et al. Cardamonin, a chalcone, inhibits human triple negative breast cancer cell invasiveness by downregulation of Wnt/beta-catenin signaling cascades and reversal of epithelial-mesenchymal transition. Biofactors 2017;43(2):152–69.
Veverka V, Crabbe T, Bird I, Lennie G, Muskett FW, Taylor RJ, et al. Structural characterization of the interaction of mTOR with phosphatidic acid and a novel class of inhibitor: compelling evidence for a central role of the FRB domain in small molecule-mediated regulation of mTOR. Oncogene 2008;27(5):585–95.
Sini P, James D, Chresta C, Guichard S. Simultaneous inhibition of mTORC1 and mTORC2 by mTOR kinase inhibitor AZD8055 induces autophagy and cell death in cancer cells. Autophagy 2010;6(4):553–4.
Zheng W, Shi D, Ji X, Han Y, Liao Q. Antiproliferation of cardamonin associated with mRNA expression of mTOR, Raptor and Rictor. Chin J Chinese Materia Med 2010;35(17):2318–23.
Dennis MD, Kimball SR, Jefferson LS. Mechanistic target of rapamycin complex 1 (mTORC1)-mediated phosphorylation is governed by competition between substrates for interaction with raptor. J Biol Chem 2013;288(1):10–9.
Matsumoto K, Onda T, Yaegashi N. Pharmacotherapy for recurrent ovarian cancer: current status and future perspectives. Jpn J Clin Oncol 2015;45(5):408–10.
Mabuchi S, Altomare DA, Cheung M, Zhang L, Poulikakos PI, Hensley HH, et al. RAD001 inhibits human ovarian cancer cell proliferation, enhances cisplatin-induced apoptosis, and prolongs survival in an ovarian cancer model. Clin Cancer Res 2007;13(14):4261–70.
Niu P, Shi D, Zhang S, Zhu Y, Zhou J. Cardamonin enhances the anti-proliferative effect of cisplatin on ovarian cancer. Oncol Lett 2018;15(3):3991–7.
Liao Q, Shi DH, Zheng W, Xu XJ, Yu YH. Antiproliferation of cardamonin is involved in mTOR on aortic smooth muscle cells in high fructose-induced insulin resistance rats. Eur J Pharmacol 2010;641(2–3):179–86.
El-Naga RN. Pre-treatment with cardamonin protects against cisplatin-induced nephrotoxicity in rats: impact on NOX-1, inflammation and apoptosis. Toxicol Appl Pharmacol 2014;274(1):87–95.
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Shi, D., Niu, P., Heng, X. et al. Autophagy induced by cardamonin is associated with mTORC1 inhibition in SKOV3 cells. Pharmacol. Rep 70, 908–916 (2018). https://doi.org/10.1016/j.pharep.2018.04.005
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DOI: https://doi.org/10.1016/j.pharep.2018.04.005