Alantolactone suppresses the metastatic phenotype and induces the apoptosis of glioblastoma cells by targeting LIMK kinase activity and activating the cofilin/G‑actin signaling cascade

Wang,Xun; Zou,Shuang; Ren,Tong; Zhao,Li-Jun; Yu,Li-Fei; Li,Xiang-Yu; Yan,Xin; Zhang,Li-Jun

doi:10.3892/ijmm.2021.4901

Alantolactone suppresses the metastatic phenotype and induces the apoptosis of glioblastoma cells by targeting LIMK kinase activity and activating the cofilin/G‑actin signaling cascade

Authors:
- Xun Wang
- Shuang Zou
- Tong Ren
- Li-Jun Zhao
- Li-Fei Yu
- Xiang-Yu Li
- Xin Yan
- Li-Jun Zhang
View Affiliations

Affiliations: Department of Neurosurgery, The Third People's Hospital of Dalian, Non‑Directly Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116033, P.R. China, Department of Neurology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310009, P.R. China, Department of Ophthalmology, The Third People's Hospital of Dalian, Non‑Directly Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116033, P.R. China, Department of Medical Oncology, The Third People's Hospital of Dalian, Non‑Directly Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116033, P.R. China
Published online on: March 1, 2021 https://doi.org/10.3892/ijmm.2021.4901
Article Number: 68
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Glioblastoma (GBM) is the most common aggressive brain tumor and is associated with an extremely poor prognosis, as the current standard of care treatments have limited efficacy. Natural compounds have attracted increasing attention as potential anticancer drugs. Alantolactone (ATL) is a natural small molecule inhibitor, that has antitumor properties. In the present study, U87MG and U251 cells were treated ATL and changes in actin/G‑actin/F‑actin/cofilin pathway were detected in whole cells, in the cytoplasm and mitochondria by western blot analysis. Immunofluorescence and immunoprecipitation analysis identified changes in the expression levels of target proteins and interactions, respectively. A LIMK enzyme inhibitor was also applied to assess the effects of ATL on the migration and invasion of GBM cells. Flow cytometry was used to detect the levels of apoptosis of GBM cells. The expression of matrix metalloproteinase (MMP)‑2/MMP‑9, caspase‑3/caspase‑9/poly(ADP‑ribose) polymerase (PARP)/cytochrome c, were determined by western blot analysis to assess the effects of targeting LIMK. The in vitro findings were verified in vivo by characterizing changes in the expression of cofilin/LIMK in xenograft tumors in immunodeficient mice. It was found that ATL activated cofilin through the targeted inhibition of LIMK enzyme activity and it thus upregulated the ratio of G/F actin, and inhibited GBM cell migration and invasion. Conversely, the activation of cofilin and G‑actin could be co‑transferred to the mitochondria to initiate the mitochondrial‑cytochrome c pathway to induce apoptosis. On the whole, the findings of the present study further illustrate the molecular mechanisms through which ATL inhibits the metastatic phenotype of GBM cells and induces apoptosis. Given previous findings, it can be deduced that ATL can function through multiple pathways and has multiple targets in GBM models, highlighting its potential for use in clinical applications.

View Figures

View References

1	Bryukhovetskiy I, Ponomarenko A, Lyakhova I, Zaitsev S, Zayats Y, Korneyko M, Eliseikina M, Mischenko P, Shevchenko V, Shanker Sharma H, et al: Personalized regulation of glioblastoma cancer stem cells based on biomedical technologies: From theory to experiment (Review). Int J Mol Med. 42:691–702. 2018.PubMed/NCBI
2	Jian S, Chen L, Minxue L, Hongmin C, Ronghua T, Xiaoxuan F, Binbin Z and Shiwen G: Tanshinone I induces apoptosis and protective autophagy in human glioblastoma cells via a reactive oxygen species-dependent pathway. Int J Mol Med. 45:983–992. 2020.PubMed/NCBI
3	Hwang TW, Kim DH, Kim DB, Jang TW, Kim GH, Moon M, Yoon KA, Choi DE, Park JH and Kim JJ: Synergistic anticancer effect of acteoside and temozolomide-based glioblastoma chemotherapy. Int J Mol Med. 43:1478–1486. 2019.PubMed/NCBI
4	Butowski NA, Sneed PK and Chang SM: Diagnosis and treatment of recurrent high-grade astrocytoma. J Clin Oncol. 24:1273–1280. 2006. View Article : Google Scholar : PubMed/NCBI
5	Cantrell CL, Abate L, Fronczek FR, Franzblau SG, Quijano L and Fischer NH: Antimycobacterial eudesmanolides from Inula helenium and Rudbeckia subtomentosa. Planta Med. 65:351–355. 1999. View Article : Google Scholar : PubMed/NCBI
6	Wang X, Yu Z, Wang C, Cheng W, Tian X, Huo X, Wang Y, Sun C, Feng L, Xing J, et al: Alantolactone, a natural sesquiterpene lactone, has potent antitumor activity against glioblastoma by targeting IKKβ kinase activity and interrupting NF-κB/COX-2-mediated signaling cascades. J Exp Clin Cancer Res. 36:932017. View Article : Google Scholar
7	Lauffenburger D and Horwitz A: Cell migration: A physically integrated molecular process. Cell. 84:359–369. 1996. View Article : Google Scholar : PubMed/NCBI
8	Mounier N and Arrigo AP: Actin cytoskeleton and small heat shock proteins: How do they interact? Cell Stress Chaperones. 7:167–176. 2002. View Article : Google Scholar : PubMed/NCBI
9	Huang T, DerMardirossian C and Bokoch G: Cofilin phosphatases and regulation of actin dynamics. Curr Opin Cell Biol. 18:26–31. 2006. View Article : Google Scholar
10	Zhang W, Gan N and Zhou J: Immunohistochemical investigation of the correlation between LIM kinase 1 expression and development and progression of human ovarian carcinoma. J Int Med Res. 40:1067–1073. 2012. View Article : Google Scholar : PubMed/NCBI
11	Zhou Y, Su J, Shi L, Liao Q and Su Q: DADS downregulates the Rac1-ROCK1/PAK1-LIMK1-ADF/cofilin signaling pathway, inhibiting cell migration and invasion. Oncol Rep. 29:605–612. 2013. View Article : Google Scholar
12	Li R, Doherty J, Antonipillai J, Chen S, Devlin M, Visser K, Baell J, Street I, Anderson RL and Bernard O: LIM kinase inhibition reduces breast cancer growth and invasiveness but systemic inhibition does not reduce metastasis in mice. Clin Exp Metastasis. 30:483–495. 2013. View Article : Google Scholar
13	Aggelou H, Chadla P, Nikou S, Karteri S, Maroulis I, Kalofonos H, Papadaki H and Bravou V: LIMK/cofilin pathway and slingshot are implicated in human colorectal cancer progression and chemoresistance. Virchows Arch. 472:727–737. 2018. View Article : Google Scholar : PubMed/NCBI
14	Karbowski M and Youle RJ: Dynamics of mitochondrial morphology in healthy cells and during apoptosis. Cell Death Differ. 10:870–880. 2003. View Article : Google Scholar : PubMed/NCBI
15	Khan M, Yi F, Rasul A, Li T, Wang N, Gao H, Gao R and Ma T: Alantolactone induces apoptosis in glioblastoma cells via GSH depletion, ROS generation, and mitochondrial dysfunction. IUBMB life. 64:783–794. 2012. View Article : Google Scholar : PubMed/NCBI
16	Chen H, Bernstein B and Bamburg J: Regulating actin-filament dynamics in vivo. Trends Biochem Sci. 25:19–23. 2000. View Article : Google Scholar : PubMed/NCBI
17	Tsai NM, Chen YL, Lee CC, Lin PC, Cheng YL, Chang WL, Lin SZ and Harn HJ: The natural compound n-butylidene- phthalide derived from angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo. J Neurochem. 99:1251–1262. 2006. View Article : Google Scholar : PubMed/NCBI
18	Li GB, Cheng Q, Liu L, Zhou T, Shan CY, Hu XY, Zhou J, Liu EH, Li P and Gao N: Mitochondrial translocation of cofilin is required for allyl isothiocyanate-mediated cell death via ROCK1/PTEN/PI3K signaling pathway. Cell Commun Signal. 11:502013. View Article : Google Scholar : PubMed/NCBI
19	Van Troys M, Huyck L, Leyman S, Dhaese S, Vandekerkhove J and Ampe C: Ins and outs of ADF/cofilin activity and regulation. Eur J Cell Biol. 87:649–667. 2008. View Article : Google Scholar : PubMed/NCBI
20	Bernstein BW and Bamburg JR: ADF/cofilin: A functional node in cell biology. Trends Cell Biol. 20:187–195. 2010. View Article : Google Scholar : PubMed/NCBI
21	Lei JC, Yu JQ, Yin Y, Liu YW and Zou GL: Alantolactone induces activation of apoptosis in human hepatoma cell. Food Chem Toxicol. 50:3313–3319. 2012. View Article : Google Scholar : PubMed/NCBI
22	Mi XG, Song ZB, Wu P, Zhang YW, Sun LG, Bao YL, Zhang Y, Zheng LH, Sun Y, Yu CL, et al: Alantolactone induces cell apoptosis partially through down-regulation of testes-specific protease 50 expression. Toxicol Lett. 224:349–355. 2014. View Article : Google Scholar
23	Shi Y, Bao YL, Wu Y, Yu CL, Huang YX, Sun Y, Zheng LH and Li YX: Alantolactone inhibits cell proliferation by interrupting the interaction between Cripto-1 and activin receptor type II A in activin signaling pathway. J Biomol Screen. 16:525–535. 2011. View Article : Google Scholar : PubMed/NCBI
24	Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT and Horwitz AR: Cell migration: Integrating signals from front to back. Science. 302:1704–1709. 2003. View Article : Google Scholar : PubMed/NCBI
25	Campellone KG and Welch MD: A nucleator arms race: Cellular control of actin assembly. Nat Rev Mol Cell Biol. 11:237–251. 2010. View Article : Google Scholar : PubMed/NCBI
26	Hitchcock-Degregori SE: Chemotaxis: Cofilin in the driver's seat. Curr Biol. 16:R1030–R1032. 2006. View Article : Google Scholar : PubMed/NCBI
27	Lee MH, Kundu JK, Chae JI and Shim JH: Targeting ROCK/LIMK/cofilin signaling pathway in cancer. Arch Pharm Res. 42:481–491. 2019. View Article : Google Scholar : PubMed/NCBI
28	Berger K and Moeller MJ: Cofilin-1 in the podocyte: A molecular switch for actin dynamics. Int Urol Nephrol. 43:273–275. 2011. View Article : Google Scholar : PubMed/NCBI
29	Wang W, Mouneimne G, Sidani M, Wyckoff J, Chen X, Makris A, Goswami S, Bresnick AR and Condeelis JS: The activity status of cofilin is directly related to invasion, intravasation, and metastasis of mammary tumors. J Cell Biol. 173:395–404. 2006. View Article : Google Scholar : PubMed/NCBI
30	Pandey D, Goyal P and Siess W: Lysophosphatidic acid stimulation of platelets rapidly induces Ca²⁺-dependent dephosphorylation of cofilin that is independent of dense granule secretion and aggregation. Blood Cells Mol Dis. 38:269–279. 2007. View Article : Google Scholar : PubMed/NCBI
31	Yang C, Yang J, Sun M, Yan J, Meng X and Ma T: Alantolactone inhibits growth of K562/adriamycin cells by downregulating Bcr/Abl and P-glycoprotein expression. IUBMB Life. 65:435–444. 2013. View Article : Google Scholar : PubMed/NCBI
32	Lei JC, Yu JQ, Yin Y, Liu YW and Zou GL: Alantolactone induces activation of apoptosis in human hepatoma cells. Food Chem Toxicol. 50:3313–3319. 2012. View Article : Google Scholar : PubMed/NCBI
33	Ding Y, Wang H, Niu J, Luo M, Gou Y, Miao L, Zou Z and Cheng Y: Induction of ROS overload by alantolactone prompts oxidative DNA damage and apoptosis in colorectal cancer cells. Int J Mol Sci. 17:5582016. View Article : Google Scholar : PubMed/NCBI
34	Pal HC, Sehar I, Bhushan S, Gupta BD and Saxena AK: Activation of caspases and poly (ADP-ribose) polymerase cleavage to induce apoptosis in leukemia HL-60 cells by Inula racemosa. Toxicol In Vitro. 24:1599–1609. 2010. View Article : Google Scholar : PubMed/NCBI
35	Dalla Via L, García-Argáez A, Martínez-Vázquez M, Grancara S, Martinis P and Toninello A: Mitochondrial permeability transition as target of anticancer drugs. Curr Pharm Des. 20:223–244. 2014. View Article : Google Scholar
36	Kanellos G and Frame MC: Cellular functions of the ADF/cofilin family at a glance. J Cell Sci. 129:3211–3218. 2016. View Article : Google Scholar : PubMed/NCBI
37	Chua BT, Volbracht C, Tan KO, Li R, Yu VC and Li P: Mitochondrial translocation of cofilin is an early step in apoptosis induction. Nat Cell Biol. 5:1083–1089. 2003. View Article : Google Scholar : PubMed/NCBI
38	Rehklau K, Gurniak CB, Conrad M, Friauf E, Ott M and Rust MB: ADF/cofilin proteins translocate to mitochondria during apoptosis but are not generally required for cell death signaling. Cell Death Differ. 19:958–967. 2012. View Article : Google Scholar :