Cucurbitacin B enhances apoptosis in gefitinib resistant non‑small cell lung cancer by modulating the miR‑17‑5p/STAT3 axis

Yu,Baodan; Zheng,Lixia; Tang,Huiqin; Wang,Weixin; Lin,Yongping

doi:10.3892/mmr.2021.12349

Cucurbitacin B enhances apoptosis in gefitinib resistant non‑small cell lung cancer by modulating the miR‑17‑5p/STAT3 axis

Authors:
- Baodan Yu
- Lixia Zheng
- Huiqin Tang
- Weixin Wang
- Yongping Lin
View Affiliations

Affiliations: Department of Laboratory Medicine, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510120, P.R. China, Department of Hematology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510120, P.R. China
Published online on: August 9, 2021 https://doi.org/10.3892/mmr.2021.12349
Article Number: 710
Copyright: © Yu 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

Tyrosine kinase inhibitors, such as gefitinib, are currently widely used as targeted therapeutics for non‑small cell lung cancer (NSCLC). Although drug resistance has become a major obstacle to successful treatment, mechanisms underlying resistance to gefitinib remain unclear. Therefore, the present study aimed to investigate the impact of adjunctive cucurbitacin B (CuB) on gefitinib resistance (GR) in the PC9 cell line, including identifying underlying mechanisms. Reverse transcription‑quantitative PCR demonstrated significant downregulation of microRNA (miR)‑17‑5p expression in GR PC9 cells (PC9/GR), and this could be reversed by CuB. During combination treatment with CuB and gefitinib at IC25, PC9/GR cell proliferation was downregulated, and apoptosis was upregulated. The presence of a miR‑17‑5p inhibitor negated the effects of CuB and gefitinib, whereas the presence of a miR‑17‑5p mimic enhanced them. Luciferase assays demonstrated that the hypothetical target gene, signal transducer and activator of transcription 3 (STAT3), was directly targeted by miR‑17‑5p. Moreover, significant elevation of the STAT3 protein and phosphorylation levels in PC9/GR cells was reversed by the addition of CuB, despite a lack of change in STAT3 transcription level. During combined treatment with CuB and gefitinib at IC25, the STAT3 protein expression was negatively associated with the expression of miR‑17‑5p. Overexpression of STAT3 increased proliferation and decreased apoptosis and the protein levels of apoptosis‑related factors cleaved caspase‑3 and cleaved caspase‑9 of PC9/GR cells. Findings indicated that STAT3 protein and phosphorylation levels became elevated in response to gefitinib, and that CuB‑induced miR‑17‑5p expression led to STAT3 degradation, thereby ameliorating GR. In summary, CuB reduced the proliferation of GR PC9 cells by modulating the miR‑17‑5p/STAT3 axis, and may represent a promising potential novel strategy for the reversal of GR.

View Figures

View References

1	Wu YL, Cheng Y, Zhou J, Lu S, Zhang Y, Zhao J, Kim DW, Soo RA, Kim SW, Pan H, et al INSIGHT Investigators, : Tepotinib plus gefitinib in patients with EGFR-mutant non-small-cell lung cancer with MET overexpression or MET amplification and acquired resistance to previous EGFR inhibitor (INSIGHT study): An open-label, phase 1b/2, multicentre, randomised trial. Lancet Respir Med. 8:1132–1143. 2020. View Article : Google Scholar : PubMed/NCBI
2	Park K, Tan EH, O'Byrne K, Zhang L, Boyer M, Mok T, Hirsh V, Yang JC, Lee KH, Lu S, et al: Afatinib versus gefitinib as first-line treatment of patients with EGFR mutation-positive non-small-cell lung cancer (LUX-Lung 7): A phase 2B, open-label, randomised controlled trial. Lancet Oncol. 17:577–589. 2016. View Article : Google Scholar : PubMed/NCBI
3	Mitsudomi T, Morita S, Yatabe Y, Negoro S, Okamoto I, Tsurutani J, Seto T, Satouchi M, Tada H, Hirashima T, et al West Japan Oncology Group, : Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): An open label, randomised phase 3 trial. Lancet Oncol. 11:121–128. 2010. View Article : Google Scholar : PubMed/NCBI
4	Lopez Sambrooks C, Baro M, Quijano A, Narayan A, Cui W, Greninger P, Egan R, Patel A, Benes CH, Saltzman WM, et al: Oligosaccharyltransferase inhibition overcomes therapeutic resistance to EGFR tyrosine kinase inhibitors. Cancer Res. 78:5094–5106. 2018. View Article : Google Scholar : PubMed/NCBI
5	Liu P, Xiang Y, Liu X, Zhang T, Yang R, Chen S, Xu L, Yu Q, Zhao H, Zhang L, et al: Cucurbitacin B induces the lysosomal degradation of EGFR and suppresses the CIP2A/PP2A/Akt signaling axis in gefitinib-resistant non-small cell lung cancer. Molecules. 24:242019.
6	Noronha V, Patil VM, Joshi A, Menon N, Chougule A, Mahajan A, Janu A, Purandare N, Kumar R, More S, et al: Gefitinib Versus Gefitinib Plus Pemetrexed and Carboplatin Chemotherapy in EGFR-Mutated Lung Cancer. J Clin Oncol. 38:124–136. 2020. View Article : Google Scholar : PubMed/NCBI
7	Xia X, Liu Y, Liao Y, Guo Z, Huang C, Zhang F, Jiang L, Wang X, Liu J and Huang H: Synergistic effects of gefitinib and thalidomide treatment on EGFR-TKI-sensitive and -resistant NSCLC. Eur J Pharmacol. 856:1724092019. View Article : Google Scholar : PubMed/NCBI
8	Li L, Jiang L, Wang Y, Zhao Y, Zhang XJ, Wu G, Zhou X, Sun J, Bai J, Ren B, et al: Combination of metformin and gefitinib as first-line therapy for nondiabetic advanced NSCLC patients with EGFR mutations: A randomized, double-blind phase II trial. Clin Cancer Res. 25:6967–6975. 2019. View Article : Google Scholar : PubMed/NCBI
9	Dandawate P, Subramaniam D, Panovich P, Standing D, Krishnamachary B, Kaushik G, Thomas SM, Dhar A, Weir SJ, Jensen RA, et al: Cucurbitacin B and I inhibits colon cancer growth by targeting the Notch signaling pathway. Sci Rep. 10:12902020. View Article : Google Scholar : PubMed/NCBI
10	Kurman Y, Kiliccioglu I, Dikmen AU, Esendagli G, Bilen CY, Sozen S and Konac E: Cucurbitacin B and cisplatin induce the cell death pathways in MB49 mouse bladder cancer model. Exp Biol Med (Maywood). 245:805–814. 2020. View Article : Google Scholar : PubMed/NCBI
11	Li Y, Wang R, Ma E, Deng Y, Wang X, Xiao J and Jing Y: The induction of G2/M cell-cycle arrest and apoptosis by cucurbitacin E is associated with increased phosphorylation of eIF2alpha in leukemia cells. Anticancer Drugs. 21:389–400. 2010. View Article : Google Scholar : PubMed/NCBI
12	Shukla S, Sinha S, Khan S, Kumar S, Singh K, Mitra K, Maurya R and Meeran SM: Cucurbitacin B inhibits the stemness and metastatic abilities of NSCLC via downregulation of canonical Wnt/β-catenin signaling axis. Sci Rep. 6:218602016. View Article : Google Scholar : PubMed/NCBI
13	Shukla S, Khan S, Kumar S, Sinha S, Farhan M, Bora HK, Maurya R, Meeran SM and Cucurbitacin B: Cucurbitacin B Alters the expression of tumor-related genes by epigenetic modifications in NSCLC and inhibits NNK-induced lung tumorigenesis. Cancer Prev Res (Phila). 8:552–562. 2015. View Article : Google Scholar : PubMed/NCBI
14	Zhang Y, Li M and Hu C: Exosomal transfer of miR-214 mediates gefitinib resistance in non-small cell lung cancer. Biochem Biophys Res Commun. 507:457–464. 2018. View Article : Google Scholar : PubMed/NCBI
15	Zhang W, Lin J, Wang P and Sun J: miR-17-5p down-regulation contributes to erlotinib resistance in non-small cell lung cancer cells. J Drug Target. 25:125–131. 2017. View Article : Google Scholar : PubMed/NCBI
16	Chen Q, Si Q, Xiao S, Xie Q, Lin J, Wang C, Chen L, Chen Q and Wang L: Prognostic significance of serum miR-17-5p in lung cancer. Med Oncol. 30:3532013. View Article : Google Scholar : PubMed/NCBI
17	Chatterjee A, Chattopadhyay D and Chakrabarti G: miR-17-5p downregulation contributes to paclitaxel resistance of lung cancer cells through altering beclin1 expression. PLoS One. 9:e957162014. View Article : Google Scholar : PubMed/NCBI
18	Yu H, Lee H, Herrmann A, Buettner R and Jove R: Revisiting STAT3 signalling in cancer: New and unexpected biological functions. Nat Rev Cancer. 14:736–746. 2014. View Article : Google Scholar : PubMed/NCBI
19	Yang Y, Wang W, Chang H, Han Z, Yu X and Zhang T: Reciprocal regulation of miR-206 and IL-6/STAT3 pathway mediates IL6-induced gefitinib resistance in EGFR-mutant lung cancer cells. J Cell Mol Med. 23:7331–7341. 2019. View Article : Google Scholar : PubMed/NCBI
20	Bartel DP: MicroRNAs: Target recognition and regulatory functions. Cell. 136:215–233. 2009. View Article : Google Scholar : PubMed/NCBI
21	Shen M, Xu Z, Xu W, Jiang K, Zhang F, Ding Q, Xu Z and Chen Y: Inhibition of ATM reverses EMT and decreases metastatic potential of cisplatin-resistant lung cancer cells through JAK/STAT3/PD-L1 pathway. J Exp Clin Cancer Res. 38:1492019. View Article : Google Scholar : PubMed/NCBI
22	Tong M, Wang J, Jiang N, Pan H and Li D: Correlation between p-STAT3 overexpression and prognosis in lung cancer: A systematic review and meta-analysis. PLoS One. 12:e01822822017. View Article : Google Scholar : PubMed/NCBI
23	Gong WJ, Ma LY, Hu L, Lv YN, Huang H, Xu JQ, Huang DD, Liu RJ, Han Y, Zhang Y, et al: STAT3 rs4796793 contributes to lung cancer risk and clinical outcomes of platinum-based chemotherapy. Int J Clin Oncol. 24:476–484. 2019. View Article : Google Scholar : PubMed/NCBI
24	Zhou J, Kwak KJ, Wu Z, Yang D, Li J, Chang M, Song Y, Zeng H, Lee LJ, Hu J, et al: PLAUR confers resistance to gefitinib through EGFR/P-AKT/survivin signaling pathway. Cell Physiol Biochem. 47:1909–1924. 2018. View Article : Google Scholar : PubMed/NCBI
25	Xu X, Li D, Liu J, Ma Z, Huang H, Min L, Dai L and Dong S: Downregulation of PTPRK promotes cell proliferation and metastasis of NSCLC by enhancing STAT3 activation. Anal Cell Pathol (Amst). 2019:42650402019.PubMed/NCBI
26	Rokavec M, Öner MG, Li H, Jackstadt R, Jiang L, Lodygin D, Kaller M, Horst D, Ziegler PK, Schwitalla S, et al: IL-6R/STAT3/miR-34a feedback loop promotes EMT-mediated colorectal cancer invasion and metastasis. J Clin Invest. 124:1853–1867. 2014. View Article : Google Scholar : PubMed/NCBI
27	Li H, Chen L, Li JJ, Zhou Q, Huang A, Liu WW, Wang K, Gao L, Qi ST and Lu YT: miR-519a enhances chemosensitivity and promotes autophagy in glioblastoma by targeting STAT3/Bcl2 signaling pathway. J Hematol Oncol. 11:702018. View Article : Google Scholar : PubMed/NCBI
28	Du W, Pan Z, Chen X, Wang L, Zhang Y, Li S, Liang H, Xu C, Zhang Y, Wu Y, et al: By targeting Stat3 microRNA-17-5p promotes cardiomyocyte apoptosis in response to ischemia followed by reperfusion. Cell Physiol Biochem. 34:955–965. 2014. View Article : Google Scholar : PubMed/NCBI
29	Liao XH, Xiang Y, Yu CX, Li JP, Li H, Nie Q, Hu P, Zhou J and Zhang TC: STAT3 is required for miR-17-5p-mediated sensitization to chemotherapy-induced apoptosis in breast cancer cells. Oncotarget. 8:15763–15774. 2017. View Article : Google Scholar : PubMed/NCBI
30	Ogino A, Kitao H, Hirano S, Uchida A, Ishiai M, Kozuki T, Takigawa N, Takata M, Kiura K and Tanimoto M: Emergence of epidermal growth factor receptor T790M mutation during chronic exposure to gefitinib in a non small cell lung cancer cell line. Cancer Res. 67:7807–7814. 2007. View Article : Google Scholar : PubMed/NCBI
31	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
32	Peng W, He D, Shan B, Wang J, Shi W, Zhao W, Peng Z, Luo Q, Duan M, Li B, et al: LINC81507 act as a competing endogenous RNA of miR-199b-5p to facilitate NSCLC proliferation and metastasis via regulating the CAV1/STAT3 pathway. Cell Death Dis. 10:5332019. View Article : Google Scholar : PubMed/NCBI
33	Liu H, Wu Z, Zhou H, Cai W, Li X, Hu J, Gao L, Feng T, Wang L, Peng X, et al: The SOX4/miR-17-92/RB1 axis promotes prostate cancer progression. Neoplasia. 21:765–776. 2019. View Article : Google Scholar : PubMed/NCBI
34	Liu F, Zhang F, Li X, Liu Q, Liu W, Song P, Qiu Z, Dong Y and Xiang H: Prognostic role of miR-17-92 family in human cancers: Evaluation of multiple prognostic outcomes. Oncotarget. 8:69125–69138. 2017. View Article : Google Scholar : PubMed/NCBI
35	Zhang X, Li Y, Qi P and Ma Z: Biology of miR-17-92 cluster and its progress in lung cancer. Int J Med Sci. 15:1443–1448. 2018. View Article : Google Scholar : PubMed/NCBI
36	Yang C, Jia X, Zhou J, Sun Q and Ma Z: The MiR-17-92 gene cluster is a blood-based marker for cancer detection in non-small-cell lung cancer. Am J Med Sci. 360:248–260. 2020. View Article : Google Scholar : PubMed/NCBI
37	Gong J, He L, Ma J, Zhang J, Wang L and Wang J: The relationship between miR-17-5p, miR-92a, and let-7b expression with non-small cell lung cancer targeted drug resistance. J BUON. 22:454–461. 2017.PubMed/NCBI
38	Friedman RC, Farh KK, Burge CB and Bartel DP: Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 19:92–105. 2009. View Article : Google Scholar : PubMed/NCBI
39	Zhou J, Liu M, Chen Y, Xu S, Guo Y and Zhao L: Cucurbitacin B suppresses proliferation of pancreatic cancer cells by ceRNA: Effect of miR-146b-5p and lncRNA-AFAP1-AS1. J Cell Physiol. 234:4655–4667. 2019. View Article : Google Scholar : PubMed/NCBI
40	Dittharot K, Dakeng S, Suebsakwong P, Suksamrarn A, Patmasiriwat P, Promkan M and Cucurbitacin B: Cucurbitacin B Induces Hypermethylation of Oncogenes in Breast Cancer Cells. Planta Med. 85:370–378. 2019. View Article : Google Scholar : PubMed/NCBI
41	Marostica LL, de Barros ALB, Oliveira J, Salgado BS, Cassali GD, Leite EA, Cardoso VN, Lang KL, Caro MS, Durán FJ, et al: Antitumor effectiveness of a combined therapy with a new cucurbitacin B derivative and paclitaxel on a human lung cancer xenograft model. Toxicol Appl Pharmacol. 329:272–281. 2017. View Article : Google Scholar : PubMed/NCBI
42	Marostica LL, Silva IT, Kratz JM, Persich L, Geller FC, Lang KL, Caro MS, Durán FJ, Schenkel EP and Simões CM: Synergistic antiproliferative effects of a new cucurbitacin B derivative and chemotherapy drugs on lung cancer cell line A549. Chem Res Toxicol. 28:1949–1960. 2015. View Article : Google Scholar : PubMed/NCBI
43	Zhang X, Sai B, Wang F, Wang L, Wang Y, Zheng L, Li G, Tang J and Xiang J: Hypoxic BMSC-derived exosomal miRNAs promote metastasis of lung cancer cells via STAT3-induced EMT. Mol Cancer. 18:402019. View Article : Google Scholar : PubMed/NCBI
44	Hu W, Ru Z, Zhou Y, Xiao W, Sun R, Zhang S, Gao Y, Li X, Zhang X and Yang H: Lung cancer-derived extracellular vesicles induced myotube atrophy and adipocyte lipolysis via the extracellular IL-6-mediated STAT3 pathway. Biochim Biophys Acta Mol Cell Biol Lipids. 1864:1091–1102. 2019. View Article : Google Scholar : PubMed/NCBI
45	Shi J, Li J, Yang S, Hu X, Chen J, Feng J, Shi T, He Y, Mei Z, He W, et al: LncRNA SNHG3 is activated by E2F1 and promotes proliferation and migration of non-small-cell lung cancer cells through activating TGF-β pathway and IL-6/JAK2/STAT3 pathway. J Cell Physiol. 235:2891–2900. 2020. View Article : Google Scholar : PubMed/NCBI