Suppression of EGFR/STAT3 activity by lupeol contributes to the induction of the apoptosis of human non‑small cell lung cancer cells

Min,Tae‑Rin; Park,Hyun‑Ji; Ha,Ki‑Tae; Chi,Gyoo‑Yong; Choi,Yung‑Hyun; Park,Shin‑Hyung

doi:10.3892/ijo.2019.4799

Suppression of EGFR/STAT3 activity by lupeol contributes to the induction of the apoptosis of human non‑small cell lung cancer cells

Authors:
- Tae‑Rin Min
- Hyun‑Ji Park
- Ki‑Tae Ha
- Gyoo‑Yong Chi
- Yung‑Hyun Choi
- Shin‑Hyung Park
View Affiliations

Affiliations: Department of Pathology, College of Korean Medicine, Dong‑Eui University, Busan 47227, Republic of Korea, Department of Korean Medical Science, School of Korean Medicine and Healthy Aging Korean Medicine Research Center, Busan National University, Yangsan, Gyeongsangnam‑do 50612, Republic of Korea, Department of Biochemistry, College of Korean Medicine, Dong‑Eui University, Busan 47227, Republic of Korea
Published online on: May 7, 2019 https://doi.org/10.3892/ijo.2019.4799
Pages: 320-330

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

The aim of this study was to investigate the underlying mechanisms responsible for the anticancer effects of lupeol on human non‑small cell lung cancer (NSCLC). MTT assay and Trypan blue exclusion assay were used to evaluate the cell viability. DAPI staining and flow cytometric analysis were used to detect apoptosis. Molecular docking and western blot analysis were performed to determine the target of lupeol. We found that lupeol suppressed the proliferation and colony formation of NSCLC cells in a dose‑dependent manner. In addition, lupeol increased chromatin condensation, poly(ADP‑ribose) polymerase (PARP) cleavage, sub‑G1 cell populations, and the proportion of Annexin V‑positive cells, indicating that lupeol triggered the apoptosis of NSCLC cells. Notably, lupeol inhibited the phosphorylation of epithelial growth factor receptor (EGFR). A docking experiment revealed that lupeol directly bound to the tyrosine kinase domain of EGFR. We observed that the signal transducer and activator of transcription 3 (STAT3), a downstream molecule of EGFR, was also dephosphorylated by lupeol. Lupeol suppressed the nuclear translocation and transcriptional activity of STAT3 and downregulated the expression of STAT3 target genes. The constitutive activation of STAT3 by STAT3 Y705D overexpression suppressed lupeol‑induced apoptosis, demonstrating that the inhibition of STAT3 activity contributed to the induction of apoptosis. The anticancer effects of lupeol were consistently observed in EGFR tyrosine kinase inhibitor (TKI)‑resistant H1975 cells (EGFR L858R/T790M). Taken together, the findings of this study suggest that lupeol may be used, not only for EGFR TKI‑naïve NSCLC, but also for advanced NSCLC with acquired resistance to EGFR TKIs.

View Figures

View References

1	Keith RL and Miller YE: Lung cancer chemoprevention: Current status and future prospects. Nat Rev Clin Oncol. 10:334–343. 2013. View Article : Google Scholar : PubMed/NCBI
2	Blackhall FH, Shepherd FA and Albain KS: Improving survival and reducing toxicity with chemotherapy in advanced non-small cell lung cancer: A realistic goal? Treat Respir Med. 4:71–84. 2005. View Article : Google Scholar
3	Travis WD, Travis LB and Devesa SS: Lung cancer. Cancer. 75(Suppl): 191–202. 1995. View Article : Google Scholar : PubMed/NCBI
4	Mosesson Y and Yarden Y: Oncogenic growth factor receptors: Implications for signal transduction therapy. Semin Cancer Biol. 14:262–270. 2004. View Article : Google Scholar : PubMed/NCBI
5	Ohsaki Y, Tanno S, Fujita Y, Toyoshima E, Fujiuchi S, Nishigaki Y, Ishida S, Nagase A, Miyokawa N, Hirata S, et al: Epidermal growth factor receptor expression correlates with poor prognosis in non-small cell lung cancer patients with p53 overexpression. Oncol Rep. 7:603–607. 2000.PubMed/NCBI
6	Inamura K, Ninomiya H, Ishikawa Y and Matsubara O: Is the epidermal growth factor receptor status in lung cancers reflected in clinicopathologic features? Arch Pathol Lab Med. 134:66–72. 2010.PubMed/NCBI
7	Siveen KS, Nguyen AH, Lee JH, Li F, Singh SS, Kumar AP, Low G, Jha S, Tergaonkar V, Ahn KS, et al: Negative regulation of signal transducer and activator of transcription-3 signalling cascade by lupeol inhibits growth and induces apoptosis in hepatocellular carcinoma cells. Br J Cancer. 111:1327–1337. 2014. View Article : Google Scholar : PubMed/NCBI
8	Scagliotti GV, Selvaggi G, Novello S and Hirsch FR: The biology of epidermal growth factor receptor in lung cancer. Clin Cancer Res. 10:S4227–S4232. 2004. View Article : Google Scholar
9	Veale D, Kerr N, Gibson GJ, Kelly PJ, Harris AL and Br J: The relationship of quantitative epidermal growth factor receptor expression in non-small cell lung cancer to long term survival. Br J Cancer. 68:162–165. 1993. View Article : Google Scholar : PubMed/NCBI
10	Fontanini G, De Laurentiis M, Vignati S, Chinè S, Lucchi M, Silvestri V, Mussi A, De Placido S, Tortora G, Bianco AR, et al: Evaluation of epidermal growth factor-related growth factors and receptors and of neoangiogenesis in completely resected stage I-IIIA non-small-cell lung cancer: Amphiregulin and microvessel count are independent prognostic indicators of survival. Clin Cancer Res. 4:241–249. 1998.PubMed/NCBI
11	Ogawa J, Iwazaki M, Inoue H, Koide S and Shohtsu A: Immunohistochemical study of glutathione-related enzymes and proliferative antigens in lung cancer. Relation to cisplatin sensitivity. Cancer. 71:2204–2209. 1993. View Article : Google Scholar : PubMed/NCBI
12	Shigematsu H and Gazdar AF: Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers. Int J Cancer. 118:257–262. 2006. View Article : Google Scholar
13	Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, et al: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 350:2129–2139. 2004. View Article : Google Scholar : PubMed/NCBI
14	Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, et al: EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science. 304:1497–1500. 2004. View Article : Google Scholar : PubMed/NCBI
15	Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I, Singh B, Heelan R, Rusch V, Fulton L, et al: EGF receptor gene mutations are common in lung cancers from 'never smokers' and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA. 101:13306–13311. 2004. View Article : Google Scholar
16	Kobayashi S, Boggon TJ, Dayaram T, Jänne PA, Kocher O, Meyerson M, Johnson BE, Eck MJ, Tenen DG and Halmos B: EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med. 352:786–792. 2005. View Article : Google Scholar : PubMed/NCBI
17	Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, Kris MG and Varmus H: Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2:e732005. View Article : Google Scholar : PubMed/NCBI
18	Johnston PA and Grandis JR: STAT3 signaling: Anticancer strategies and challenges. Mol Interv. 11:18–26. 2011. View Article : Google Scholar : PubMed/NCBI
19	Harada D, Takigawa N and Kiura K: The role of STAT3 in non-small cell lung cancer. Cancers (Basel). 6:708–722. 2014. View Article : Google Scholar
20	Jiang R, Jin Z, Liu Z, Sun L, Wang L and Li K: Correlation of activated STAT3 expression with clinicopathologic features in lung adenocarcinoma and squamous cell carcinoma. Mol Diagn Ther. 15:347–352. 2011. View Article : Google Scholar
21	Xu YH and Lu S: A meta-analysis of STAT3 and phospho-STAT3 expression and survival of patients with non-small-cell lung cancer. Eur J Surg Oncol. 40:311–317. 2014. View Article : Google Scholar
22	Gao SP, Chang Q, Mao N, Daly LA, Vogel R, Chan T, Liu SH, Bournazou E, Schori E, Zhang H, et al: JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors. Sci Signal. 9:ra332016. View Article : Google Scholar : PubMed/NCBI
23	Yao Z, Fenoglio S, Gao DC, Camiolo M, Stiles B, Lindsted T, Schlederer M, Johns C, Altorki N, Mittal V, et al: TGF-beta IL-6 axis mediates selective and adaptive mechanisms of resistance to molecular targeted therapy in lung cancer. Proc Natl Acad Sci USA. 107:15535–15540. 2010. View Article : Google Scholar : PubMed/NCBI
24	Li R, Hu Z, Sun SY, Chen ZG, Owonikoko TK, Sica GL, Ramalingam SS, Curran WJ, Khuri FR and Deng X: Niclosamide overcomes acquired resistance to erlotinib through suppression of STAT3 in non-small cell lung cancer. Mol Cancer Ther. 12:2200–2212. 2013. View Article : Google Scholar : PubMed/NCBI
25	Jung MJ, Rho JK, Kim YM, Jung JE, Jin YB, Ko YG, Lee JS, Lee SJ, Lee JC and Park MJ: Upregulation of CXCR4 is functionally crucial for maintenance of stemness in drug-resistant non-small cell lung cancer cells. Oncogene. 32:209–221. 2013. View Article : Google Scholar
26	Fan W, Tang Z, Yin L, Morrison B, Hafez-Khayyata S, Fu P, Huang H, Bagai R, Jiang S, Kresak A, et al: MET-independent lung cancer cells evading EGFR kinase inhibitors are therapeutically susceptible to BH3 mimetic agents. Cancer Res. 71:4494–4505. 2011. View Article : Google Scholar : PubMed/NCBI
27	Zulkifli AA, Tan FH, Putoczki TL, Stylli SS and Luwor RB: STAT3 signaling mediates tumour resistance to EGFR targeted therapeutics. Mol Cell Endocrinol. 451:15–23. 2017. View Article : Google Scholar : PubMed/NCBI
28	Siddique HR and Saleem M: Beneficial health effects of lupeol triterpene: A review of preclinical studies. Life Sci. 88:285–293. 2011. View Article : Google Scholar
29	Wu XT, Liu JQ, Lu XT, Chen FX, Zhou ZH, Wang T, Zhu SP and Fei SJ: The enhanced effect of lupeol on the destruction of gastric cancer cells by NK cells. Int Immunopharmacol. 16:332–340. 2013. View Article : Google Scholar : PubMed/NCBI
30	Liu Y, Bi T, Dai W, Wang G, Qian L, Shen G and Gao Q: Lupeol enhances inhibitory effect of 5-fluorouracil on human gastric carcinoma cells. Naunyn Schmiedebergs Arch Pharmacol. 389:477–484. 2016. View Article : Google Scholar : PubMed/NCBI
31	Tarapore RS, Siddiqui IA, Adhami VM, Spiegelman VS and Mukhtar H: The dietary terpene lupeol targets colorectal cancer cells with constitutively active Wnt/β-catenin signaling. Mol Nutr Food Res. 57:1950–1958. 2013. View Article : Google Scholar : PubMed/NCBI
32	Pitchai D, Roy A and Ignatius C: In vitro evaluation of anticancer potentials of lupeol isolated from Elephantopus scaber L. on MCF-7 cell line. J Adv Pharm Technol Res. 5:179–184. 2014. View Article : Google Scholar : PubMed/NCBI
33	Sankaranarayanan S, Bama P, Sathyabama S and Bhuvaneswari N: Anticancer compound isolated from the leaves of Tridax procumbens against human lung cancer cell A-549. Asian J Pharm Clin Res. 6:91–96. 2013.
34	He W, Li X and Xia S: Lupeol triterpene exhibits potent antitumor effects in A427 human lung carcinoma cells via mitochondrial mediated apoptosis, ROS generation, loss of mitochondrial membrane potential and downregulation of m-TOR/PI3Ksol;AKT signalling pathway. J BUON. 23:635–640. 2018.PubMed/NCBI
35	Lee TK, Castilho A, Cheung VC, Tang KH, Ma S and Ng IO: Lupeol targets liver tumor-initiating cells through phosphatase and tensin homolog modulation. Hepatology. 53:160–170. 2011. View Article : Google Scholar
36	Liu Y, Bi T, Shen G, Li Z, Wu G, Wang Z, Qian L and Gao Q: Lupeol induces apoptosis and inhibits invasion in gallbladder carcinoma GBC-SD cells by suppression of EGFR/MMP-9 signaling pathway. Cytotechnology. 68:123–133. 2016. View Article : Google Scholar :
37	Rauth S, Ray S, Bhattacharyya S, Mehrotra DG, Alam N, Mondal G, Nath P, Roy A, Biswas J and Murmu N: Lupeol evokes anticancer effects in oral squamous cell carcinoma by inhibiting oncogenic EGFR pathway. Mol Cell Biochem. 417:97–110. 2016. View Article : Google Scholar : PubMed/NCBI
38	Grosdidier A, Zoete V and Michielin O: SwissDock, a protein-small molecule docking web service based on EADock DSS. Nucleic Acids Res. 39(Suppl): W270–7. 2011. View Article : Google Scholar : PubMed/NCBI
39	Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN and Bourne PE: The protein data bank. Nucleic Acids Res. 28:235–242. 2000. View Article : Google Scholar
40	Choowongkomon K, Sawatdichaikul O, Songtawee N and Limtrakul J: Receptor-based virtual screening of EGFR kinase inhibitors from the NCI diversity database. Molecules. 15:4041–4054. 2010. View Article : Google Scholar : PubMed/NCBI
41	Stamos J, Sliwkowski MX and Eigenbrot C: Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor. J Biol Chem. 277:46265–46272. 2002. View Article : Google Scholar : PubMed/NCBI