Claudin‑1 silencing increases sensitivity of liver cancer HepG2 cells to 5‑fluorouracil by inhibiting autophagy

Tong,Hui; Li,Tao; Qiu,Weihua; Zhu,Zhecheng

doi:10.3892/ol.2019.10967

Claudin‑1 silencing increases sensitivity of liver cancer HepG2 cells to 5‑fluorouracil by inhibiting autophagy

Authors:
- Hui Tong
- Tao Li
- Weihua Qiu
- Zhecheng Zhu
View Affiliations

Affiliations: Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China
Published online on: October 8, 2019 https://doi.org/10.3892/ol.2019.10967
Pages: 5709-5716
Copyright: © Tong 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

Liver cancer is one of the most common cancer types globally. However, the acquisition of drug resistance limits the effectiveness of chemotherapy and commonly results in metastasis. Therefore, an effective therapeutic approach to target chemoresistance‑associated cellular molecules is imperative. Claudin‑1 (CLDN1) has previously been reported to be associated with the development of drug resistance. The present study investigated the effect of CLDN1 on the sensitivity of 5‑fluorouracil (5‑FU)‑resistant liver cancer cells. Firstly, a 5‑FU‑resistant HepG2 liver cancer cell line (Hep/5FU) was developed by continuous 5‑FU treatment. MTT proliferation, Transwell and Matrigel assays indicated that Hep/5FU cells were significantly resistant to 5‑FU, and demonstrated increased migration and invasion abilities, compared with parental HepG2 cells. Furthermore, reverse transcription‑quantitative polymerase chain reaction and western blot analysis indicated that mRNA and protein expression levels of CLDN1 were significantly increased in Hep/5FU cells, compared with HepG2 cells. CLDN1 was knocked down by transfection with small interference RNA. MTT and Annexin V‑fluorescein isothiocyanate/propidium iodide assays demonstrated that CLDN1 silencing significantly inhibits proliferation and enhances apoptosis induced by 5‑FU treatment in Hep/5FU cells, compared with non‑silenced Hep/5FU cells. Additionally, CLDN1 silencing attenuated the migration and invasion capabilities of Hep/5FU cells. In addition, it was identified that CLDN1 silencing decreased drug resistance by inhibiting autophagy, which was associated with a decrease in the ratio of microtubule‑associated protein 1A/1B‑light chain 3 (LC3)‑II/LC3‑I and upregulation of P62. A cell proliferation assay revealed that the addition of autophagy inhibitor 3‑methyladenine decreased drug resistance of Hep/5FU cells. By contrast, incubation with the autophagy agonist Rapamycin elevated drug resistance of CLDN1‑silenced Hep/5FU cells. In summary, these data indicate that CLDN1 may be a potential target for resensitizing resistant liver cancer HepG2 cells to 5‑FU by regulating cell autophagy.

View Figures

View References

1	American Cancer SocietyCancer Facts & Figures 2014. Atlanta: American Cancer Society; 2014
2	Krishna R and Mayer LD: Multidrug resistance (MDR) in cancer: Mechanisms, reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs. Eur J Pharm Sci. 11:265–283. 2000. View Article : Google Scholar : PubMed/NCBI
3	Deng GL, Zeng S and Shen H: Chemotherapy and target therapy for hepatocellular carcinoma: New advances and challenges. World J Hepatol. 7:787–798. 2015. View Article : Google Scholar : PubMed/NCBI
4	Petraccia L, Onori P, Sferra R, Lucchetta MC, Liberati G, Grassi M and Gaudio E: MDR (multidrug resistance) in hepatocarcinoma clinical-therapeutic implications. Clin Ter. 154:325–335. 2003.(In Italian). PubMed/NCBI
5	Longley DB, Harkin DP and Johnston PG: 5-fluorouracil: Mechanisms of action and clinical strategies. Nat Rev Cancer. 3:330–338. 2003. View Article : Google Scholar : PubMed/NCBI
6	Ohtsu A: Chemotherapy for metastatic gastric cancer: Past, present, and future. J Gastroenterol. 43:256–264. 2008. View Article : Google Scholar : PubMed/NCBI
7	Obert E, Strauss R, Brandon C, Grek C, Ghatnekar G, Gourdie R and Rohrer B: Targeting the tight junction protein, zonula occludens-1, with the connexin43 mimetic peptide, aCT1, reduces VEGF-dependent RPE pathophysiology. J Mol Med (Berl). 95:535–552. 2017. View Article : Google Scholar : PubMed/NCBI
8	Matsuoka H, Shima A, Uda A, Ezaki H and Michihara A: The retinoic acid receptor-related orphan receptor a positively regulates tight junction protein claudin domain-containing 1 mRNA expression in human brain endothelial cells. J Biochem. 161:441–450. 2017.PubMed/NCBI
9	Liu W, Mi S, Ruan Z, Li J, Shu X, Yao K, Jiang M and Deng Z: Dietary tryptophan enhanced the expression of tight junction protein ZO-1 in intestine. J Food Sci. 82:562–567. 2017. View Article : Google Scholar : PubMed/NCBI
10	Morita K, Furuse M, Fujimoto K and Tsukita S: Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc Natl Acad Sci USA. 96:511–516. 1999. View Article : Google Scholar : PubMed/NCBI
11	Furuse M, Fujita K, Hiiragi T, Fujimoto K and Tsukita S: Claudin-1 and-2: Novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol. 141:1539–1550. 1998. View Article : Google Scholar : PubMed/NCBI
12	Kim B and Breton S: The MAPK/ERK-signaling pathway regulates the expression and distribution of tight junction proteins in the mouse proximal epididymis. Biol Reprod. 94:222016. View Article : Google Scholar : PubMed/NCBI
13	Akizuki R, Shimobaba S, Matsunaga T, Endo S and Ikari A: Claudin-5, −7 and −18 suppress proliferation mediated by inhibition of phosphorylation of Akt in human lung squamous cell carcinoma. Biochim Biophys Acta. 1864:293–302. 2017. View Article : Google Scholar
14	Yang Y, Cheon S, Jung MK, Song SB, Kim D, Kim HJ, Park H, Bang SI and Cho D: Interleukin-18 enhances breast cancer cell migration via down-regulation of claudin-12 and induction of the p38 MAPK pathway. Biochem Biophys Res Commun. 459:379–386. 2015. View Article : Google Scholar : PubMed/NCBI
15	Zhang WN, Li W, Wang XL, Hu Z, Zhu D, Ding WC, Liu D, Li KZ, Ma D and Wang H: CLDN1 expression in cervical cancer cells is related to tumor invasion and metastasis. Oncotarget. 7:87449–87461. 2016. View Article : Google Scholar : PubMed/NCBI
16	Kuo KT, Chen CL, Chou TY, Yeh CT, Lee WH and Wang LS: Nm23H1 mediates tumor invasion in esophageal squamous cell carcinoma by regulation of CLDN1 through the AKT signaling. Oncogenesis. 5:e2392016. View Article : Google Scholar : PubMed/NCBI
17	Kominsky SL: Claudins: Emerging targets for cancer therapy. Expert Rev Mol Med. 8:1–11. 2006. View Article : Google Scholar : PubMed/NCBI
18	Moldvay J, Fábián K, Jäckel M, Németh Z, Bogos K, Furák J, Tiszlavicz L, Fillinger J, Döme B and Schaff Z: Claudin-1 protein expression is a good prognostic factor in non-small cell lung cancer, but only in squamous cell carcinoma cases. Pathol Oncol Res. 23:151–56. 2017. View Article : Google Scholar : PubMed/NCBI
19	Sun BS, Yao YQ, Pei BX, Zhang ZF and Wang CL: Claudin-1 correlates with poor prognosis in lung adenocarcinoma. Thorac Cancer. 7:556–563. 2016. View Article : Google Scholar : PubMed/NCBI
20	Meena AS, Sharma A, Kumari R, Mohammad N, Singh SV and Bhat MK: Inherent and acquired resistance to paclitaxel in hepatocellular carcinoma: Molecular events involved. PLoS One. 8:e615242013. View Article : Google Scholar : PubMed/NCBI
21	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
22	Vu NB, Nguyen TT, Tran LC, Do CD, Nguyen BH, Phan NK and Pham PV: Doxorubicin and 5-fluorouracil resistant hepatic cancer cells demonstrate stem-like properties. Cytotechnology. 65:491–503. 2013. View Article : Google Scholar : PubMed/NCBI
23	Tolba MF and Abdel-Rahman SZ: Pterostilbine, an active component of blueberries, sensitizes colon cancer cells to 5-fluorouracil cytotoxicity. Sci Rep. 5:152392015. View Article : Google Scholar : PubMed/NCBI
24	Nakagawa S, Miyoshi N, Ishii H, Mimori K, Tanaka F, Sekimoto M, Doki Y and Mori M: Expression of CLDN1 in colorectal cancer: A novel marker for prognosis. Int J Oncol. 39:791–967. 2011.PubMed/NCBI
25	Jian Y, Chen C, Li B and Tian X: Delocalized Claudin-1 promotes metastasis of human osteosarcoma cells. Biochem Biophys Res Commun. 466:356–361. 2015. View Article : Google Scholar : PubMed/NCBI
26	Zhao X, Zou Y, Gu Q, Zhao G, Gray H, Pfeffer LM and Yue J: Lentiviral vector mediated Claudin1 silencing inhibits epithelial to mesenchymal transition in breast cancer cells. Viruses. 7:2965–2979. 2015. View Article : Google Scholar : PubMed/NCBI
27	Fortier AM, Asselin E and Cadrin M: Keratin 8 and 18 loss in epithelial cancer cells increases collective cell migration and cisplatin sensitivity through claudin1 up-regulation. J Biol Chem. 288:11555–7129. 2013. View Article : Google Scholar : PubMed/NCBI
28	Lee JW, Hsiao WT, Chen HY, Hsu LP, Chen PR, Lin MD, Chiu SJ, Shih WL and Hsu YC: Upregulated claudin-1 expression confers resistance to cell death of nasopharyngeal carcinoma cells. Int J Cancer. 126:1353–1366. 2010.PubMed/NCBI
29	Kim H, Kim Y and Jeoung D: DDX53 promotes cancer stem cell-like properties and autophagy. Mol Cells. 40:54–65. 2017. View Article : Google Scholar : PubMed/NCBI
30	Lee YJ and Jang BK: The role of autophagy in hepatocellular carcinoma. Int J Mol Sci. 16:26629–26643. 2015. View Article : Google Scholar : PubMed/NCBI
31	Jang JE, Eom JI, Jeung HK, Cheong JW, Lee JY, Kim JS and Min YH: Targeting AMPK-ULK1-mediated autophagy for combating BET inhibitor resistance in acute myeloid leukemia stem cells. Autophagy. 13:761–762. 2017. View Article : Google Scholar : PubMed/NCBI
32	Nie T, Yang S, Ma H, Zhang L, Lu F, Tao K, Wang R, Yang R, Huang L, Mao Z and Yang Q: Regulation of ER stress-induced autophagy by GSK3b-TIP60-ULK1 pathway. Cell Death Dis. 7:e25632016. View Article : Google Scholar : PubMed/NCBI
33	Kim J, Kundu M, Viollet B and Guan KL: AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol. 13:132–141. 2011. View Article : Google Scholar : PubMed/NCBI
34	Mercer CA, Kaliappan A and Dennis PB: A novel, human Atg13 binding protein, Atg101, interacts with ULK1 and is essential for macroautophagy. Autophagy. 5:649–662. 2009. View Article : Google Scholar : PubMed/NCBI