Generic placeholder image

Current Cancer Drug Targets

Author(s): Gaojie Song, Chao Shang, Yilong Zhu, Zhiru Xiu, Yaru Li, Xia Yang, Chenchen Ge, Jicheng Han, Ningyi Jin, Yiquan Li*, Xiao Li* and Jinbo Fang*

DOI: 10.2174/1568009623666221025150239

DownloadDownload PDF Flyer Cite As
Apoptin Inhibits Glycolysis and Regulates Autophagy by Targeting Pyruvate Kinase M2 (PKM2) in Lung Cancer A549 Cells

Page: [411 - 424] Pages: 14

  • * (Excluding Mailing and Handling)

Abstract

Background: Pyruvate kinase M2 (PKM2) is a key enzyme in aerobic glycolysis and plays an important role in tumor energy metabolism and tumor growth. Ad-apoptin, a recombinant oncolytic adenovirus, can stably express apoptin in tumor cells and selectively causes cell death in tumor cells.

Objective: The relationship between the anti-tumor function of apoptin, including apoptosis and autophagy activation, and the energy metabolism of tumor cells has not been clarified.

Methods: In this study, we used the A549 lung cancer cell line to analyze the mechanism of PKM2 involvement in apoptin-mediated cell death in tumor cells. PKM2 expression in lung cancer cells was detected by Western blot and qRT-PCR. In the PKM2 knockdown and over-expression experiments, A549 lung cancer cells were treated with Ad-apoptin, and cell viability was determined by the CCK-8 assay and crystal violet staining. Glycolysis was investigated using glucose consumption and lactate production experiments. Moreover, the effects of Ad-apoptin on autophagy and apoptosis were analyzed by immunofluorescence using the Annexin v-mCherry staining and by western blot for c-PARP, p62, and LC3-II proteins. Immunoprecipitation analysis was used to investigate the interaction between apoptin and PKM2. In addition, following PKM2 knockdown and overexpression, the expression levels of p-AMPK, p-mTOR, p-ULK1, and p-4E-BP1 proteins in Ad-apoptin treated tumor cells were analyzed by western blot to investigate the mechanism of apoptin effect on the energy metabolism of tumor cells. The in vivo antitumor mechanism of apoptin was analyzed by xenograft tumor inhibition experiment in nude mice and immunohistochemistry of tumors’ tissue.

Results: As a result, apoptin could target PKM2, inhibit glycolysis and cell proliferation in A549 cells, and promote autophagy and apoptosis in A549 cells by regulating the PKM2/AMPK/mTOR pathway.

Conclusion: This study confirmed the necessary role of Ad-apoptin in the energy metabolism of A549 cells.

Graphical Abstract

[1]
Vander Heiden, M.G.; Cantley, L.C.; Thompson, C.B. Understanding the Warburg effect: The metabolic requirements of cell proliferation. Science, 2009, 324(5930), 1029-1033.
[http://dx.doi.org/10.1126/science.1160809] [PMID: 19460998]
[2]
Zhu, H.; Luo, H.; Zhu, X.; Hu, X.; Zheng, L.; Zhu, X. Pyruvate kinase M2 (PKM2) expression correlates with prognosis in solid cancers: A meta-analysis. Oncotarget, 2017, 8(1), 1628-1640.
[http://dx.doi.org/10.18632/oncotarget.13703] [PMID: 27911861]
[3]
Wong, N.; Ojo, D.; Yan, J.; Tang, D. PKM2 contributes to cancer metabolism. Cancer Lett., 2015, 356(2), 184-191.
[http://dx.doi.org/10.1016/j.canlet.2014.01.031] [PMID: 24508027]
[4]
Shi, H.; Li, D.; Zhang, J.; Wang, Y.; Yang, L.; Zhang, H.; Wang, X.; Mu, B.; Wang, W.; Ma, Y.; Guo, F.; Wei, Y. Silencing of pkm2 in-creases the efficacy of docetaxel in human lung cancer xenografts in mice. Cancer Sci., 2010, 101(6), 1447-1453.
[http://dx.doi.org/10.1111/j.1349-7006.2010.01562.x] [PMID: 20507318]
[5]
Wang, C.; Jiang, J.; Ji, J.; Cai, Q.; Chen, X.; Yu, Y.; Zhu, Z.; Zhang, J. PKM2 promotes cell migration and inhibits autophagy by mediating PI3K/AKT activation and contributes to the malignant development of gastric cancer. Sci. Rep., 2017, 7(1), 2886.
[http://dx.doi.org/10.1038/s41598-017-03031-1] [PMID: 28588255]
[6]
Lim, J.Y.; Yoon, S.O.; Seol, S.Y.; Hong, S.W.; Kim, J.W.; Choi, S.H.; Cho, J.Y. Overexpression of the M2 isoform of pyruvate kinase is an adverse prognostic factor for signet ring cell gastric cancer. World J. Gastroenterol., 2012, 18(30), 4037-4043.
[http://dx.doi.org/10.3748/wjg.v18.i30.4037] [PMID: 22912555]
[7]
Chen, Z.; Lu, X.; Wang, Z.; Jin, G.; Wang, Q.; Chen, D.; Chen, T.; Li, J.; Fan, J.; Cong, W.; Gao, Q.; He, X. Co-expression of PKM2 and TRIM35 predicts survival and recurrence in hepatocellular carcinoma. Oncotarget, 2015, 6(4), 2539-2548.
[http://dx.doi.org/10.18632/oncotarget.2991] [PMID: 25576919]
[8]
Zhao, H.; Song, L. TKP, a serine protease from trichosanthes kirilowii, inhibits cell proliferation by blocking aerobic glycolysis in hepato-cellular carcinoma cells. Nutr. Cancer, 2022, 74(1), 333-345.
[9]
Wen, Y.Y.; Liu, W.T.; Sun, H.R.; Ge, X.; Shi, Z.M.; Wang, M.; Li, W.; Zhang, J.Y.; Liu, L.Z.; Jiang, B.H. IGF-1-mediated PKM2/β-catenin/miR-152 regulatory circuit in breast cancer. Sci. Rep., 2017, 7(1), 15897.
[http://dx.doi.org/10.1038/s41598-017-15607-y] [PMID: 29162853]
[10]
Guo, M.; Zhao, X.; Yuan, X.; Jiang, J.; Li, P. MiR-let-7a inhibits cell proliferation, migration, and invasion by down-regulating PKM2 in cervical cancer. Oncotarget, 2017, 8(17), 28226-28236.
[http://dx.doi.org/10.18632/oncotarget.15999] [PMID: 28415668]
[11]
Lang, N.; Wang, C.; Zhao, J.; Shi, F.; Wu, T.; Cao, H. Long non coding RNA BCYRN1 promotes glycolysis and tumor progression by regulating the miR 149/PKM2 axis in non small cell lung cancer. Mol. Med. Rep., 2020, 21(3), 1509-1516.
[http://dx.doi.org/10.3892/mmr.2020.10944] [PMID: 32016455]
[12]
Hong, B.; Zhu, Q.; Zhang, L.; Wang, J. Pyruvate kinase M2 inhibits the progression of bladder cancer by targeting MAKP pathway. J. Cancer Res. Ther., 2018, 14(10)(Suppl.), 616.
[http://dx.doi.org/10.4103/0973-1482.187302] [PMID: 30249877]
[13]
Zhang, X.; Liu, T.; Zheng, S.; Liu, Q.; Shen, T.; Han, X.; Zhang, Q.; Yang, L.; Lu, X. SUMOylation of HSP27 regulates PKM2 to promote esophageal squamous cell carcinoma progression. Oncol. Rep., 2020, 44(4), 1355-1364.
[http://dx.doi.org/10.3892/or.2020.7711] [PMID: 32945483]
[14]
He, C.L.; Bian, Y.Y.; Xue, Y.; Liu, Z.X.; Zhou, K.Q.; Yao, C.F.; Lin, Y.; Zou, H.F.; Luo, F.X.; Qu, Y.Y.; Zhao, J.Y.; Ye, M.L.; Zhao, S.M.; Xu, W. Pyruvate kinase M2 activates mTORC1 by phosphorylating AKT1S1. Sci. Rep., 2016, 6(1), 21524.
[http://dx.doi.org/10.1038/srep21524] [PMID: 26876154]
[15]
Chu, B.; Wang, J.; Wang, Y.; Yang, G. Knockdown of PKM2 induces apoptosis and autophagy in human A549 alveolar adenocarcinoma cells. Mol. Med. Rep., 2015, 12(3), 4358-4363.
[http://dx.doi.org/10.3892/mmr.2015.3943] [PMID: 26082202]
[16]
Zhu, Y.; Li, Y.; Bai, B.; Shang, C.; Fang, J.; Cong, J.; Li, W.; Li, S.; Song, G.; Liu, Z.; Zhao, J.; Li, X.; Zhu, G.; Jin, N. Effects of apoptin-induced endoplasmic reticulum stress on lipid metabolism, migration, and invasion of HepG-2 cells. Front. Oncol., 2021, 11, 614082.
[http://dx.doi.org/10.3389/fonc.2021.614082] [PMID: 33718168]
[17]
Noteborn, M.H.M. Proteins selectively killing tumor cells. Eur. J. Pharmacol., 2009, 625(1-3), 165-173.
[http://dx.doi.org/10.1016/j.ejphar.2009.06.068] [PMID: 19836376]
[18]
Zhuang, S.M.; Shvarts, A.; van Ormondt, H.; Jochemsen, A.G.; van der Eb, A.J.; Noteborn, M.H. Apoptin, a protein derived from chicken anemia virus, induces p53-independent apoptosis in human osteosarcoma cells. Cancer Res., 1995, 55(3), 486-489.
[PMID: 7834613]
[19]
Zhang, Y.; Leliveld, S.R.; Kooistra, K.; Molenaar, C.; Rohn, J.L.; Tanke, H.J.; Abrahams, J.P.; Noteborn, M.H. Recombinant apoptin mul-timers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion. Exp. Cell Res., 2003, 289(1), 36-46.
[http://dx.doi.org/10.1016/S0014-4827(03)00188-5] [PMID: 12941602]
[20]
Li, X.; Jin, N.; Mi, Z.; Lian, H.; Sun, L.; Li, X.; Zheng, H. Antitumor effects of a recombinant fowlpox virus expressing Apoptin in vivo and in vitro. Int. J. Cancer, 2006, 119(12), 2948-2957.
[http://dx.doi.org/10.1002/ijc.22215] [PMID: 17036330]
[21]
Xiao, L.; Yan, L.; Zhongmei, W.; Chang, L.; Huijun, L.; Mingyao, T.; Kuoshi, J.; Lili, S.; Pegn, G.; Encheng, Y.; Xiaohong, X.; Shifu, K.; Zhuoyue, W.; Yuhang, W.; Ningyi, J. Potent anti-tumor effects of a dual specific oncolytic adenovirus expressing apoptin in vitro and in vivo. Mol. Cancer, 2010, 9(1), 10.
[http://dx.doi.org/10.1186/1476-4598-9-10] [PMID: 20085660]
[22]
Chen, S.; Li, Y.Q.; Yin, X.Z.; Li, S.Z.; Zhu, Y.L.; Fan, Y.Y.; Li, W.J.; Cui, Y.L.; Zhao, J.; Li, X.; Zhang, Q.G.; Jin, N.Y. Recombinant ade-noviruses expressing apoptin suppress the growth of MCF 7 breast cancer cells and affect cell autophagy. Oncol. Rep., 2019, 41(5), 2818-2832.
[http://dx.doi.org/10.3892/or.2019.7077] [PMID: 30896879]
[23]
Song, G.; Fang, J.; Shang, C.; Li, Y.; Zhu, Y.; Xiu, Z.; Sun, L.; Jin, N.; Li, X. Ad-apoptin inhibits glycolysis, migration and invasion in lung cancer cells targeting AMPK/mTOR signaling pathway. Exp. Cell Res., 2021, 409(2), 112926.
[http://dx.doi.org/10.1016/j.yexcr.2021.112926] [PMID: 34793774]
[24]
Kimura, S.; Noda, T.; Yoshimori, T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy, 2007, 3(5), 452-460.
[http://dx.doi.org/10.4161/auto.4451] [PMID: 17534139]
[25]
Inoki, K.; Kim, J.; Guan, K.L. AMPK and mTOR in cellular energy homeostasis and drug targets. Annu. Rev. Pharmacol. Toxicol., 2012, 52(1), 381-400.
[http://dx.doi.org/10.1146/annurev-pharmtox-010611-134537] [PMID: 22017684]
[26]
Xu, X.; Kong, X.; Liu, T.; Zhou, L.; Wu, J.; Fu, J.; Wang, Y.; Zhu, M.; Yao, S.; Ding, Y.; Ding, L.; Li, R.; Zhu, X.; Tang, X.; Zhang, Y.; Yang, Q.; Ling, J.; Zhou, H. Metastasis-associated protein 1, modulated by miR-30c, promotes endometrial cancer progression through AKT/mTOR/4E-BP1 pathway. Gynecol. Oncol., 2019, 154(1), 207-217.
[http://dx.doi.org/10.1016/j.ygyno.2019.04.005] [PMID: 30979588]
[27]
Duma, N.; Santana-Davila, R.; Molina, J.R. Non–small cell lung cancer: Epidemiology, screening, diagnosis, and treatment. Mayo Clin. Proc., 2019, 94(8), 1623-1640.
[http://dx.doi.org/10.1016/j.mayocp.2019.01.013] [PMID: 31378236]
[28]
Tai, G.; Zhang, M.; Liu, F. Circ_0000735 enhances the proliferation, metastasis and glycolysis of non-small cell lung cancer by regulating the miR-635/FAM83F axis. Exp. Lung Res., 2021, 47(3), 1-13.
[http://dx.doi.org/10.1080/01902148.2021.1881188] [PMID: 33560141]
[29]
Yan, D.; Chen, Y. Tumor mutation burden (TMB)-associated signature constructed to predict survival of lung squamous cell carcinoma patients. Sci. Rep., 2021, 11(1), 9020.
[http://dx.doi.org/10.1038/s41598-021-88694-7] [PMID: 33907270]
[30]
Jie, L.; Cong, L.; Conghui, W.; Ying, G. GTPBP2 positively regulates the invasion, migration and proliferation of non-small cell lung can-cer. J. Cancer, 2021, 12(13), 3819-3826.
[http://dx.doi.org/10.7150/jca.48340] [PMID: 34093790]
[31]
Simeone, J.C.; Nordstrom, B.L.; Patel, K.; Klein, A.B. Treatment patterns and overall survival in metastatic non-small-cell lung cancer in a real-world, US setting. Future Oncol., 2019, 15(30), 3491-3502.
[http://dx.doi.org/10.2217/fon-2019-0348] [PMID: 31497994]
[32]
Cui, C.; Li, Y.; Sun, Y.; Zhu, Y.; Fang, J.; Bai, B. Antitumor effect of a dual cancer-specific oncolytic adenovirus on prostate cancer PC-3 cells. Urol. Oncol., 2019, 37, e1-e18.
[33]
Li, W.; Li, Y.; Cui, Y.; Li, S.; Zhu, Y.; Shang, C.; Song, G.; Liu, Z.; Xiu, Z.; Cong, J.; Li, T.; Li, X.; Sun, L.; Jin, N. Anti-tumour effects of a dual cancer‐specific oncolytic adenovirus on Breast Cancer Stem cells. J. Cell. Mol. Med., 2021, 25(2), 666-676.
[http://dx.doi.org/10.1111/jcmm.16113] [PMID: 33305893]
[34]
Liu, X.; Yang, Z.; Li, Y.; Zhu, Y.; Li, W.; Li, S.; Wang, J.; Cui, Y.; Shang, C.; Liu, Z.; Song, G.; Li, C.; Li, X.; Shao, G.; Jin, N. Chemoviro-therapy of lung squamous cell carcinoma by combining oncolytic adenovirus with gemcitabine. Front. Oncol., 2020, 10, 229.
[http://dx.doi.org/10.3389/fonc.2020.00229] [PMID: 32158698]
[35]
Li, Y.; Zhu, Y.; Fang, J.; Li, W.; Li, S.; Liu, X.; Liu, Z.; Song, G.; Shang, C.; Cong, J.; Bai, B.; Sun, L.; Jin, N.; Li, X. Apoptin regulates apoptosis and autophagy by modulating Reactive Oxygen Species (ROS) levels in human liver cancer cells. Front. Oncol., 2020, 10, 1026.
[http://dx.doi.org/10.3389/fonc.2020.01026] [PMID: 32714864]
[36]
Yang, G.; Meng, X.; Sun, L.; Hu, N.; Jiang, S.; Sheng, Y.; Chen, Z.; Zhou, Y.; Chen, D.; Li, X.; Jin, N. Antitumor effects of a dual cancer-specific oncolytic adenovirus on colorectal cancer in vitro and in vivo. Exp. Ther. Med., 2015, 9(2), 327-334.
[http://dx.doi.org/10.3892/etm.2014.2086] [PMID: 25574193]
[37]
Akashi, S.; Nishida, T.; Mizukawa, T.; Kawata, K.; Takigawa, M.; Iida, S.; Kubota, S. Regulation of cellular communication network factor 2 (CCN2) in breast cancer cells via the cell-type dependent interplay between CCN2 and glycolysis. J. Oral Biosci./ JAOB, Jpn. Assoc. Oral Biol., 2020, 62(3), 280-288.
[http://dx.doi.org/10.1016/j.job.2020.07.001] [PMID: 32791309]
[38]
Xia, Y.; Kang, T.W.; Jung, Y.D.; Zhang, C.; Lian, S. Sulforaphane inhibits nonmuscle invasive bladder cancer cells proliferation through suppression of HIF-1α-mediated glycolysis in hypoxia. J. Agric. Food Chem., 2019, 67(28), 7844-7854.
[http://dx.doi.org/10.1021/acs.jafc.9b03027] [PMID: 31241937]
[39]
Fang, G.; Zhang, P.; Liu, J.; Zhang, X.; Zhu, X.; Li, R.; Wang, H. Inhibition of GSK-3β activity suppresses HCC malignant phenotype by inhibiting glycolysis via activating AMPK/mTOR signaling. Cancer Lett., 2019, 463, 11-26.
[http://dx.doi.org/10.1016/j.canlet.2019.08.003] [PMID: 31404613]
[40]
He, X.; Du, S.; Lei, T.; Li, X.; Liu, Y.; Wang, H.; Tong, R.; Wang, Y. PKM2 in carcinogenesis and oncotherapy. Oncotarget, 2017, 8(66), 110656-110670.
[http://dx.doi.org/10.18632/oncotarget.22529] [PMID: 29299177]
[41]
Fan, F.T.; Shen, C.S.; Tao, L.; Tian, C.; Liu, Z.G.; Zhu, Z.J.; Liu, Y.P.; Pei, C.S.; Wu, H.Y.; Zhang, L.; Wang, A.Y.; Zheng, S.Z.; Huang, S.L.; Lu, Y. PKM2 regulates hepatocellular carcinoma cell epithelial-mesenchymal transition and migration upon EGFR activation. Asian Pac. J. Cancer Prev., 2014, 15(5), 1961-1970.
[http://dx.doi.org/10.7314/APJCP.2014.15.5.1961] [PMID: 24716919]
[42]
Li, R.Z.; Fan, X.X.; Shi, D.F.; Zhu, G.Y.; Wang, Y.W.; Luo, L.X.; Pan, H.D.; Yao, X.J.; Leung, E.L.H.; Liu, L. Identification of a new py-ruvate kinase M2 isoform (PKM2) activator for the treatment of non-small-cell lung cancer (NSCLC). Chem. Biol. Drug Des., 2018, 92(5), 1851-1858.
[http://dx.doi.org/10.1111/cbdd.13354] [PMID: 29931766]
[43]
Barroso-Chinea, P.; Luis-Ravelo, D.; Fumagallo-Reading, F.; Castro-Hernandez, J.; Salas-Hernandez, J.; Rodriguez-Nuñez, J.; Febles-Casquero, A.; Cruz-Muros, I.; Afonso-Oramas, D.; Abreu-Gonzalez, P.; Moratalla, R.; Millan, M.J.; Gonzalez-Hernandez, T. DRD3 (do-pamine receptor D3) but not DRD2 activates autophagy through MTORC1 inhibition preserving protein synthesis. Autophagy, 2020, 16(7), 1279-1295.
[http://dx.doi.org/10.1080/15548627.2019.1668606] [PMID: 31538542]
[44]
Roberson, P.A.; Shimkus, K.L.; Welles, J.E.; Xu, D.; Whitsell, A.L.; Kimball, E.M.; Jefferson, L.S.; Kimball, S.R. A time course for mark-ers of protein synthesis and degradation with hindlimb unloading and the accompanying anabolic resistance to refeeding. J. Appl. Physiol., 2020, 129(1), 36-46.
[http://dx.doi.org/10.1152/japplphysiol.00155.2020] [PMID: 32407240]
[45]
Steiner, J.L.; Lang, C.H. Ethanol acutely antagonizes the refeeding-induced increase in mTOR-dependent protein synthesis and decrease in autophagy in skeletal muscle. Mol. Cell. Biochem., 2019, 456(1-2), 41-51.
[http://dx.doi.org/10.1007/s11010-018-3488-4] [PMID: 30523512]