Warfarin affects the proliferation and apoptosis of lung cancer cells by regulating the Gas6/Axl/PI3K/Akt/NF-κB pathway

doi:10.5246/jcps.2023.03.016

Abstract

Abstract:

The antitumor potential of warfarin is demonstrated in different experimental cancer model systems. However, the mechanism of warfarin in reducing the incidence of lung cancer is unclear. In the present study, we aimed to investigate the effect and the possible mechanism of warfarin on the proliferation and apoptosis of lung cancer cell line H226. It was an in vitro experiment. The lung cancer cell line H226 was intervened with warfarin at the concentrations of 128, 256, and 512 μmol/L. The cell proliferation was determined by CCK8 assay, and the cell apoptosis was evaluated by flow cytometry and Western blotting analysis. The effects of warfarin on Gas6/Axl/PI3K/Akt/NF-κB protein and gene expression were determined by Western blotting analysis and RT-PCR, respectively. Warfarin inhibited the growth of H226 cells at 24, 48, and 72 h, showing a dose- and time-dependent manner. Flow cytometry results showed that the apoptosis rate of H226 cells was significantly increased when treated with 128, 256, and 512 μmol/L warfarin. Meanwhile, the expression of Bcl-2 protein was decreased, and the expressions of Bax protein and Caspase 3 protein were increased, showing significant differences compared with the blank group (P < 0.05). Compared with the control group, warfarin and Axl inhibitor BGB324 could inhibit the protein expression of the Gas6/Axl pathway and its downstream pathway (P < 0.05) significantly and decrease the gene expression of the Gas6/Axl/PI3K/Akt/NF-κB pathway significantly (P < 0.05). Warfarin could inhibit the proliferation and induce the apoptosis of the lung cell line. The possible mechanism might be related to the inhibitory effect on Gas6/Axl/PI3K/Akt/NF-κB pathway proteins and genes.

Key words: Warfarin, Lung neoplasms, Cell proliferation, Apoptosis, In vitro techniques

Supporting:

Limei Yang, Liman Wang, Xuhui Huang, Jie Zhuang. Warfarin affects the proliferation and apoptosis of lung cancer cells by regulating the Gas6/Axl/PI3K/Akt/NF-κB pathway[J]. Journal of Chinese Pharmaceutical Sciences, 2023, 32(3): 190-199.

Figures/Tables 7

Table 1. The sequences of primer for RT-PCR.

Table 2. Effects of warfarin on gene expressions of Gas6/Axl/PI3K/Akt/NF-κB pathway genes (n = 3).

References 25

[1]	Semakula, J.R.; Kisa, G.; Mouton, J.P.; Cohen, K.; Blockman, M.; Pirmohamed, M.; Sekaggya-Wiltshire, C.; Waitt, C. Anticoagulation in sub-Saharan Africa: are direct oral anticoagulants the answer? A review of lessons learnt from warfarin. Br. J. Clin. Pharmacol. 2021, 87, 3699–3705.
[2]	Haaland, G.S.; Falk, R.S.; Straume, O.; Lorens, J.B. Association of warfarin use with lower overall cancer incidence among patients older than 50 years. JAMA Intern. Med. 2017, 177, 1774–1780.
[3]	Remková, A.; Remko, M. May warfarin prevent cancer? Vnitr. Lek. 2019, 65, 679–684.
[4]	Chen, X.J.; Jin, D.Y.; Stafford, D.W.; Tie, J.K. Evaluation of oral anticoagulants with vitamin K epoxide reductase in its native milieu. Blood. 2018, 132, 1974–1984.
[5]	Kirane, A.; Ludwig, K.F.; Sorrelle, N.; Haaland, G.; Sandal, T.; Ranaweera, R.; Toombs, J.E.; Wang, M.; Dineen, S.P.; Micklem, D.; Dellinger, M.T.; Lorens, J.B.; Brekken, R.A. Warfarin blocks Gas6-mediated axl activation required for pancreatic cancer epithelial plasticity and metastasis. Cancer Res. 2015, 75, 3699–3705.
[6]	Su, S.C.; Chiang, C.F.; Hsieh, C.H.; Lu, G.H.; Liu, J.S.; Shieh, Y.S.; Hung, Y.J.; Lee, C.H. Growth arrest-specific 6 modulates adiponectin expression and insulin resistance in adipose tissue. J. Diabetes Investig. 2021, 12, 485–492.
[7]	Pilli, V.S.; Datta, A.; Dorsey, A.; Liu, B.; Majumder, R. Modulation of protein S and growth arrest specific 6 protein signaling inhibits pancreatic cancer cell survival and proliferation. Oncol. Rep. 2020, 44, 1322–1332.
[8]	Cui, L.W.; Bai, Y.L.; Zhang, J.X.; Zhang, S.L.; Xu, J.S. Effects of extracellular acid stimulation on rat vascular smooth muscle cell in Gas6/Axl or PI3K/Akt signaling pathway. Clin. Exp. Hypertens. 2016, 38, 451–456.
[9]	Paccez, J.D.; Vasques, G.J.; Correa, R.G.; Vasconcellos, J.F.; Duncan, K.; Gu, X.; Bhasin, M.; Libermann, T.A.; Zerbini, L.F. The receptor tyrosine kinase Axl is an essential regulator of prostate cancer proliferation and tumor growth and represents a new therapeutic target. Oncogene. 2013, 32, 689–698.
[10]	Shinh, Y.S.; Lai, C.Y.; Kao, Y.R.; Shiah, S.G.; Chu, Y.W.; Lee, H.S.; Wu, C.W. Expression of axl in lung adenocarcinoma and correlation with tumor progression. Neoplasia. 2005, 7, 1058–1064.
[11]	Melaragno, M.G.; Cavet, M.E.; Yan, C.; Tai, L.K.; Jin, Z.G.; Haendeler, J.; Berk, B.C. Gas6 inhibits apoptosis in vascular smooth muscle: role of Axl kinase and Akt. J. Mol. Cell Cardiol. 2004, 37, 881–887.
[12]	Cunningham, M.S.; Preston, R.J.S.; O’Donnell, J.S. Does antithrombotic therapy improve survival in cancer patients? Blood Rev. 2009, 23, 129–135.
[13]	Wu, Y.S.; Yang, L.M.; Huang, X.H.; Huang, F.; Han, T. Influence of CYP2C93 and CYP4F2 rs2108622 gene polymorphisms on over-anticoagulation and bleeding complications of warfari n therapy in Chinese patients: a cohort study. J. Chin. Pharm. Sci. 2021*, 6, 484–494.
[14]	Zacharski, L.R.; Prandoni, P.; Monreal, M. Warfarin versus low-molecular-weight heparin therapy in cancer patients. Oncologist. 2005, 10, 72–79.
[15]	Spitz, A.Z.; Gavathiotis, E. Physiological and pharmacological modulation of BAX. Trends Pharmacol. Sci. 2022, 43, 206–220.
[16]	Dhani, S.; Zhao, Y.; Zhivotovsky, B. A long way to go: caspase inhibitors in clinical use. Cell Death Dis. 2021, 12, 949.
[17]	D’Orsi, B.; Mateyka, J.; Prehn, J.H.M. Control of mitochondrial physiology and cell death by the Bcl-2 family proteins Bax and Bok. Neurochem. Int. 2017, 109, 162–170.
[18]	Asadi, M.; Taghizadeh, S.; Kaviani, E.; Vakili, O.; Taheri-Anganeh, M.; Tahamtan, M.; Savardashtaki, A. Caspase-3: structure, function, and biotechnological aspects. Biotechnol. Appl. Biochem. 2022, 69, 1633–1645.
[19]	Gay, C.M.; Balaji, K.; Byers, L.A. Giving AXL the axe: targeting AXL in human malignancy. Br. J. Cancer. 2017, 116, 415–423.
[20]	Sun, Z.G.; Liu, J.H.; Zhang, JM.; Qian, Y. Research Progress of Axl Inhibitors. Curr. Top. Med. Chem. 2019, 19, 1338–1349.
[21]	Wimmel, A.; Glitz, D.; Kraus, A.; Roeder, J.; Schuermann, M. Axl receptor tyrosine kinase expression in human lung cancer cell lines correlates with cellular adhesion. Eur. J. Cancer. 2001, 37, 2264–2274.
[22]	Shinh, Y.S.; Lai, C.Y.; Kao, Y.R.; Shiah, S.G.; Chu, Y.W.; Lee, H.S.; Wu, C.W. Expression of axl in lung adenocarcinoma and correlation with tumor progression. Neoplasia. 2005, 7, 1058–1064.
[23]	Ishikawa, M.; Sonobe, M.; Nakayama, E.; Kobayashi, M.; Kikuchi, R.; Kitamura, J.; Imamura, N.; Date, H. Higher expression of receptor tyrosine kinase Axl, and differential expression of its ligand, Gas6, predict poor survival in lung adenocarcinoma patients. Ann. Surg. Oncol. 2013, 20, S467–S476.
[24]	Paccez, J.D.; Vasques, G.J.; Correa, R.G.; Vasconcellos, J.F.; Duncan, K.; Gu, X.; Bhasin, M.; Libermann, T.A.; Zerbini, L.F. The receptor tyrosine kinase Axl is an essential regulator of prostate cancer proliferation and tumor growth and represents a new therapeutic target. Oncogene. 2013, 32, 689–698.
[25]	Kanzaki, R.; Naito, H.; Kise, K.; Takara, K.; Eino, D.; Minami, M.; Shintani, Y.; Funaki, S.; Kawamura, T.; Kimura, T.; Okumura, M.; Takakura, N. Gas6 derived from cancer-associated fibroblasts promotes migration of Axl-expressing lung cancer cells during chemotherapy. Sci. Rep. 2017, 7, 10613.