p21 promotes gemcitabine tolerance in A549 cells by inhibiting DNA damage and altering the cell cycle

Fu,Tian; Ma,Xuan; Du,Shen-Lin; Ke,Zhi-Yin; Wang,Xue-Chun; Yin,Hai-Han; Wang,Wen-Xuan; Liu,Yong-Jun; Liang,Ai-Ling

doi:10.3892/ol.2023.14059

p21 promotes gemcitabine tolerance in A549 cells by inhibiting DNA damage and altering the cell cycle

Authors:
- Tian Fu
- Xuan Ma
- Shen-Lin Du
- Zhi-Yin Ke
- Xue-Chun Wang
- Hai-Han Yin
- Wen-Xuan Wang
- Yong-Jun Liu
- Ai-Ling Liang
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Affiliations: Department of Biochemistry and Molecular Biology, Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical University, Dongguan, Guangdong 523808, P.R. China, Department of Clinical Laboratory, Xinle City Hospital, Shijiazhuang, Hebei 050700, P.R. China, Department of Clinical Laboratory, Dongguan People's Hospital, Dongguan, Guangdong 523058, P.R. China
Published online on: September 20, 2023 https://doi.org/10.3892/ol.2023.14059
Article Number: 471
Copyright: © Fu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Gemcitabine is one of the most widely used chemotherapy drugs for advanced malignant tumors, including non‑small cell lung cancer. However, the clinical efficacy of gemcitabine is limited due to drug resistance. The aim of the present study was to investigate the role of p21 in gemcitabine‑resistant A549 (A549/G⁺) lung cancer cells. IC₅₀ values were determined using a Cell Counting Kit‑8 (CCK‑8) assay. mRNA and protein expression levels of genes were measured by reverse transcription‑quantitative PCR and western blotting, respectively. The cell cycle distribution and apoptosis rate were analyzed by flow cytometry. DNA damage in cells was evaluated by single‑cell gel electrophoresis. The results of western blot analysis and the CCK‑8 assay demonstrated that the expression of p21 was higher in A549/G⁺ cells than in gemcitabine‑sensitive cells. Knockdown of p21 expression in gemcitabine‑resistant cells sensitized these cells to gemcitabine (with the IC₅₀ decreasing from 84.2 to 26.7 µM). Cell cycle analysis revealed different changes in the cell cycle distribution in A549/G⁺ cells treated with the same concentration of gemcitabine, and decreased expression of p21 was shown to promote G₁ arrest. The apoptosis assay and comet assay results revealed that decreased p21 expression resulted in accumulation of unrepaired DNA double‑strand breaks (DSBs) and induction of apoptosis by gemcitabine. The present study demonstrated that knockout of p21 mRNA expression in A549/G⁺ cells promotes apoptosis and DNA DSB accumulation, accompanied by G₁ arrest. These results indicated that p21 is involved in regulating the response of A549 cells to gemcitabine.

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1	Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A and Bray F: Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 71:209–249. 2021. View Article : Google Scholar : PubMed/NCBI
2	He L, Luo L, Zhu H, Yang H, Zhang Y, Wu H, Sun H, Jiang F, Kathera CS, Liu L, et al: FEN1 promotes tumor progression and confers cisplatin resistance in non-small-cell lung cancer. Mol Oncol. 11:640–654. 2017. View Article : Google Scholar : PubMed/NCBI
3	Zhang X, Wang D, Li Z, Jiao D, Jin L, Cong J, Zheng X and Xu L: Low-Dose gemcitabine treatment enhances immunogenicity and natural killer cell-driven tumor immunity in lung cancer. Front Immunol. 11:3312020. View Article : Google Scholar : PubMed/NCBI
4	Toschi L, Finocchiaro G, Bartolini S, Gioia V and Cappuzzo F: Role of gemcitabine in cancer therapy. Future Oncol. 1:7–17. 2005. View Article : Google Scholar : PubMed/NCBI
5	Mini E, Nobili S, Caciagli B, Landini I and Mazzei T: Cellular pharmacology of gemcitabine. Ann Oncol. 17 (Suppl 5):v7–v12. 2006. View Article : Google Scholar : PubMed/NCBI
6	Li Y, Wang LR, Chen J, Lou Y and Zhang GB: First-line gemcitabine plus cisplatin in nonsmall cell lung cancer patients. Dis Markers. 2014:9604582014. View Article : Google Scholar : PubMed/NCBI
7	Jones RM, Kotsantis P, Stewart GS, Groth P and Petermann E: BRCA2 and RAD51 promote double-strand break formation and cell death in response to gemcitabine. Mol Cancer Ther. 13:2412–2421. 2014. View Article : Google Scholar : PubMed/NCBI
8	de Sousa Cavalcante L and Monteiro G: Gemcitabine: Metabolism and molecular mechanisms of action, sensitivity and chemoresistance in pancreatic cancer. Eur J Pharmacol. 741:8–16. 2014. View Article : Google Scholar : PubMed/NCBI
9	Liu JS, Yeh CA, Huang IC, Huang GY, Chiu CH, Mahalakshmi B, Wen SY, Huang CY and Kuo WW: Signal transducer and activator of transcription 3 mediates apoptosis inhibition through reducing mitochondrial ROS and activating Bcl-2 in gemcitabine-resistant lung cancer A549 cells. J Cell Physiol. 236:3896–3905. 2021. View Article : Google Scholar : PubMed/NCBI
10	Deng T, Yan G, Song X, Xie L, Zhou Y, Li J, Hu X, Li Z, Hu J, Zhang Y, et al: Deubiquitylation and stabilization of p21 by USP11 is critical for cell-cycle progression and DNA damage responses. Proc Natl Acad Sci USA. 115:4678–4683. 2018. View Article : Google Scholar : PubMed/NCBI
11	Zlotorynski E: Cancer biology: The dark side of p21. Nat Rev Mol Cell Biol. 17:4612016. View Article : Google Scholar : PubMed/NCBI
12	Warfel NA and El-Deiry WS: p21WAF1 and tumourigenesis: 20 years after. Curr Opin Oncol. 25:52–58. 2013. View Article : Google Scholar : PubMed/NCBI
13	Georgakilas AG, Martin OA and Bonner WM: p21: A two-faced genome guardian. Trends Mol Med. 23:310–319. 2017. View Article : Google Scholar : PubMed/NCBI
14	Wang H, Zhu LJ, Yang YC, Wang ZX and Wang R: MiR-224 promotes the chemoresistance of human lung adenocarcinoma cells to cisplatin via regulating G₁/S transition and apoptosis by targeting p21^WAF1/CIP1. Br J Cancer. 111:339–354. 2014. View Article : Google Scholar : PubMed/NCBI
15	Liu Z, Sun M, Lu K, Liu J, Zhang M, Wu W, De W, Wang Z and Wang R: The long noncoding RNA HOTAIR contributes to cisplatin resistance of human lung adenocarcinoma cells via downregualtion of p21(WAF1/CIP1) expression. PLoS One. 8:e772932013. View Article : Google Scholar : PubMed/NCBI
16	Zhao J, Fu W, Liao H, Dai L, Jiang Z, Pan Y, Huang H, Mo Y, Li S, Yang G and Yin J: The regulatory and predictive functions of miR-17 and miR-92 families on cisplatin resistance of non-small cell lung cancer. BMC Cancer. 15:7312015. View Article : Google Scholar : PubMed/NCBI
17	Guo Q, Lan F, Yan X, Xiao Z, Wu Y and Zhang Q: Hypoxia exposure induced cisplatin resistance partially via activating p53 and hypoxia inducible factor-1α in non-small cell lung cancer A549 cells. Oncol Lett. 16:801–808. 2018.PubMed/NCBI
18	Liang AL, Du SL, Zhang B, Zhang J, Ma X, Wu CY and Liu YJ: Screening miRNAs associated with resistance gemcitabine from exosomes in A549 lung cancer cells. Cancer Manag Res. 11:6311–6321. 2019. View Article : Google Scholar : PubMed/NCBI
19	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
20	Ma X, Fu T, Ke ZY, Du SL, Wang XC, Zhou N, Zhong MY, Liu YJ and Liang AL: MiR-17-5p/RRM2 regulated gemcitabine resistance in lung cancer A549 cells. Cell Cycle. 22:1367–1379. 2023. View Article : Google Scholar : PubMed/NCBI
21	Galanos P, Vougas K, Walter D, Polyzos A, Maya-Mendoza A, Haagensen EJ, Kokkalis A, Roumelioti FM, Gagos S, Tzetis M, et al: Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing. Nat Cell Biol. 18:777–789. 2016. View Article : Google Scholar : PubMed/NCBI
22	Zohny SF, Al-Malki AL, Zamzami MA and Choudhry H: p21^Waf1/Cip1: Its paradoxical effect in the regulation of breast cancer. Breast Cancer. 26:131–137. 2019. View Article : Google Scholar : PubMed/NCBI
23	Zamagni A, Pasini A, Pirini F, Ravaioli S, Giordano E, Tesei A, Calistri D, Ulivi P, Fabbri F, Foca F, et al: CDKN1A upregulation and cisplatin-pemetrexed resistance in non-small cell lung cancer cells. Int J Oncol. 56:1574–1584. 2020.PubMed/NCBI
24	Ewald B, Sampath D and Plunkett W: H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation. Mol Cancer Ther. 6:1239–1248. 2007. View Article : Google Scholar : PubMed/NCBI
25	Price JG, Idoyaga J, Salmon H, Hogstad B, Bigarella CL, Ghaffari S, Leboeuf M and Merad M: CDKN1A regulates Langerhans cell survival and promotes Treg cell generation upon exposure to ionizing irradiation. Nat Immunol. 16:1060–1068. 2015. View Article : Google Scholar : PubMed/NCBI
26	Karimian A, Ahmadi Y and Yousefi B: Multiple functions of p21 in cell cycle, apoptosis and transcriptional regulation after DNA damage. DNA Repair (Amst). 42:63–71. 2016. View Article : Google Scholar : PubMed/NCBI
27	Jana S, Patra K, Jana J, Mandal DP and Bhattacharjee S: Nrf-2 transcriptionally activates P21^Cip/WAF1 and promotes A549cell survival against oxidative stress induced by H₂O₂. Chem Biol Interact. 285:59–68. 2018. View Article : Google Scholar : PubMed/NCBI
28	Xia X, Ma Q, Li X, Ji T, Chen P, Xu H, Li K, Fang Y, Weng D, Weng Y, et al: Cytoplasmic p21 is a potential predictor for cisplatin sensitivity in ovarian cancer. BMC Cancer. 11:3992011. View Article : Google Scholar : PubMed/NCBI
29	Koster R, di Pietro A, Timmer-Bosscha H, Gibcus JH, van den Berg A, Suurmeijer AJ, Bischoff R, Gietema JA and de Jong S: Cytoplasmic p21 expression levels determine cisplatin resistance in human testicular cancer. J Clin Invest. 120:3594–3605. 2010. View Article : Google Scholar : PubMed/NCBI
30	Maiuthed A, Ninsontia C, Erlenbach-Wuensch K, Ndreshkjana B, Muenzner JK, Caliskan A, Husayn AP, Chaotham C, Hartmann A, Vial Roehe A, et al: Cytoplasmic p21 mediates 5-fluorouracil resistance by inhibiting pro-apoptotic Chk2. Cancers (Basel). 10:3732018. View Article : Google Scholar : PubMed/NCBI
31	Zhao WS, Yan WP, Chen DB, Dai L, Yang YB, Kang XZ, Fu H, Chen P, Deng KJ, Wang XY, et al: Genome-scale CRISPR activation screening identifies a role of ELAVL2-CDKN1A axis in paclitaxel resistance in esophageal squamous cell carcinoma. Am J Cancer Res. 9:1183–1200. 2019.PubMed/NCBI
32	Ikeda R, Vermeulen LC, Lau E, Jiang Z, Sachidanandam K, Yamada K and Kolesar JM: Isolation and characterization of gemcitabine-resistant human non-small cell lung cancer A549 cells. Int J Oncol. 38:513–519. 2011.PubMed/NCBI
33	Gaben AM, Saucier C, Bedin M, Barbu V and Mester J: Rapamycin inhibits cdk4 activation, p 21(WAF1/CIP1) expression and G1-phase progression in transformed mouse fibroblasts. Int J Cancer. 108:200–206. 2004. View Article : Google Scholar : PubMed/NCBI
34	Dijk SN, Protasoni M, Elpidorou M, Kroon AM and Taanman JW: Mitochondria as target to inhibit proliferation and induce apoptosis of cancer cells: The effects of doxycycline and gemcitabine. Sci Rep. 10:43632020. View Article : Google Scholar : PubMed/NCBI
35	Santarelli R, Pompili C, Gilardini Montani MS, Romeo MA, Gonnella R, D'Orazi G and Cirone M: Lovastatin reduces PEL cell survival by phosphorylating ERK1/2 that blocks the autophagic flux and engages a cross-talk with p53 to activate p21. IUBMB Life. 73:968–977. 2021. View Article : Google Scholar : PubMed/NCBI
36	Tay VSY, Devaraj S, Koh T, Ke G, Crasta KC and Ali Y: Increased double strand breaks in diabetic β-cells with a p21 response that limits apoptosis. Sci Rep. 9:193412019. View Article : Google Scholar : PubMed/NCBI
37	Wettersten HI, Hee Hwang S, Li C, Li C, Shiu EY, Wecksler AT, Hammock BD and Weiss RH: A novel p21 attenuator which is structurally related to sorafenib. Cancer Biol Ther. 14:278–285. 2013. View Article : Google Scholar : PubMed/NCBI
38	Yan G, Dawood M, Böckers M, Klauck SM, Fottner C, Weber MM and Efferth T: Multiple modes of cell death in neuroendocrine tumors induced by artesunate. Phytomedicine. 79:1533322020. View Article : Google Scholar : PubMed/NCBI
39	Minagawa Y, Ishino K, Wada R, Kudo M, Naito Z, Takeshita T and Ohashi R: High Expression of p21 as a potential therapeutic target in ovarian clear-cell carcinoma. Anticancer Res. 40:5631–5639. 2020. View Article : Google Scholar : PubMed/NCBI