IL‑6 plays a crucial role in epithelial‑mesenchymal transition and pro‑metastasis induced by sorafenib in liver cancer - PubMed (original) (raw)

IL‑6 plays a crucial role in epithelial‑mesenchymal transition and pro‑metastasis induced by sorafenib in liver cancer

Ke-Wei Zhang et al. Oncol Rep. 2021 Mar.

Abstract

Interleukin‑6 (IL‑6) is involved in various biological responses, including tumor progression, metastasis and chemoresistance. However, the role and molecular mechanism of IL‑6 in the treatment of sorafenib in liver cancer remain unclear. In the present study, through western blot analysis, Transwell assay, flow cytometric assay, ELISA analysis and immunohistochemistry it was revealed that sorafenib promoted metastasis and induced epithelial‑mesenchymal transition (EMT) in liver cancer cells in vitro and in vivo, and significantly increased IL‑6 expression. Endogenous or exogenous IL‑6 affected metastasis and EMT progression in liver cancer cells through Janus kinase 2/signal transducer and activator of transcription 3 (STAT3) signaling. Knocked out IL‑6 markedly attenuated the pro‑metastasis effect of sorafenib and increased the susceptibility of liver cancer cells to it. In conclusion, the present results indicated that IL‑6/STAT3 signaling may be a novel therapeutic strategy for liver cancer.

Keywords: liver cancer; interleukin-6; sorafenib; drug resistance; epithelial-mesenchymal transition.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.

Figure 1.

Sorafenib increases the metastatic potential of liver cancer cells and IL-6 knockout attenuates the pro-invasive effect induced by the treatment of sorafenib. (A and B) Transwell assays revealed that sorafenib increased the metastatic potential of HCCLM3-wt and HepG2-wt cells (both ***P<0.001). (C and D) IL-6 expression was disrupted by TALEN. Stably transfected clones were validated through RT-qPCR and western blot analysis (both ****P<0.0001). (E) Transwell assays revealed that IL-6 knockout attenuated the pro-invasive effect induced by sorafenib in HCCLM3-IL-6(−) (P>0.05). HCC, hepatocellular carcinoma; IL-6, interleukin-6; wt, wild-type; TALEN, transcription activator-like effector nucleases; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Figure 2.

Figure 2.

Sorafenib decreases the tumor volume and increases the intrahepatic metastatic potential and lung metastatic potential of liver cancer cells in vivo. IL-6 knockout attenuated the pro-invasive effect induced by the treatment of sorafenib in vivo. (A and B) Sorafenib decreased the tumor volume in the HCCLM3-wt and HCCLM3-IL-6(−) groups (both *P<0.05). (C and D) Sorafenib increased the number of IHMs in the HCCLM3-wt sorafenib group compared with the HCCLM3-wt control, while sorafenib did not increase the number of IHMs in the HCCLM3-IL-6(−) sorafenib group compared with the HCCLM3-IL-6(−) control. (**P<0.01 and P>0.05, respectively). The number of IHMs in the HCCLM3-wt control was not higher than that in the HCCLM3-IL-6(−) control (P>0.05). (E and F) Sorafenib increased the number of lung metastases in the HCCLM3-wt sorafenib group compared with the HCCLM3-wt control, while sorafenib did not increase the number of lung metastases in the HCCLM3-IL-6(−) sorafenib group compared with the HCCLM3-IL-6(−) control. (****P<0.0001 and P>0.05, respectively). The number of lung metastases in the HCCLM3-wt control was higher than that in the HCCLM3-IL-6(−) control (****P<0.0001). IL-6, interleukin-6; IHMs, intrahepatic metastases; HCC, hepatocellular carcinoma; wt, wild-type.

Figure 3.

Figure 3.

Sorafenib induces EMT and upregulates IL-6 in HCCLM3-wt and HepG2-wt cells, as revealed by western blot analysis. (A and B) E-cadherin was downregulated, and N-cadherin, vimentin and Snail were upregulated by sorafenib in HCCLM3-wt and HepG2-wt cells (all ****P<0.0001). (C and D) Sorafenib upregulated IL-6 in HCCLM3-wt and HepG2-wt cells (both ****P<0.0001). EMT, epithelial-mesenchymal transition; IL-6, interleukin-6; HCC, hepatocellular carcinoma; wt, wild-type.

Figure 4.

Figure 4.

IL-6 knockout inhibits tumor cell growth, as revealed by CCK-8 assay, flow cytometric analysis and western blot analysis. (A) CCK-8 assay for cell proliferation of HCCLM3-wt and HCCLM3-IL-6(−) cells. IL-6 knockout inhibited liver cancer cell proliferation, as revealed by CCK-8 assay (***P<0.001). (B) Flow cytometric cycle assay of HCCLM3-wt and HCCLM3-IL-6(−) cells revealed that the knockout of IL-6 increased the proportion of cells at the G1 phase and decreased that of cells in the S phase (both *P<0.05). (C) Flow cytometric apoptosis assay of HCCLM3-wt and HCCLM3-IL-6(−) cells revealed that the knockout of IL-6 increased the cell apoptosis ratio as indicated by western blot analysis (*P<0.05). (D) Western blot analysis revealed that anti-apoptotic marker (Bcl-2) and cell cycle markers (cyclin D1 and CDK2) were downregulated in HCCLM3-IL-6(−) cells, as compared with HCCLM3-wt cells, whereas pro-apoptotic markers cleaved caspase-3 and cleaved PARP were upregulated in HCCLM3-IL-6(−) cells (all ****P<0.0001). CCK-8, Cell Counting Kit-8; wt, wild-type; IL-6, interleukin-6; HCC, hepatocellular carcinoma; Bcl-2, B-cell lymphoma-2.

Figure 5.

Figure 5.

The knockout of IL-6 decreases the metastatic ability of HCCLM3 cells, and exogenous IL-6 increases the metastasis ability of HepG2 cells, as revealed by Transwell assay and western blot analysis. (A) The knockout of IL-6 decreased the metastatic potential of HCCLM3-wt cells, as revealed by Transwell assay (***P<0.001). (B) IL-6 knockout upregulated E-cadherin, and downregulated N-cadherin, vimentin and Snail in HCCLM3-IL-6(−) cells, as compared with HCCLM3-wt cells (all ****P<0.0001). (C) Exogenous IL-6 increased the metastatic ability of HepG2-wt cells as revealed by Transwell assay (**P<0.01). (D) Exogenous IL-6 downregulated E-cadherin, and upregulated N-cadherin, vimentin and Snail in HepG2-wt cells (all ****P<0.0001). IL-6, interleukin-6; HCC, hepatocellular carcinoma; wt, wild-type.

Figure 6.

Figure 6.

IL-6 knockout increases the susceptivity of HCCLM3 cells to sorafenib, as revealed by CCK-8 assay, flow cytometric analysis and western blot analysis. (A and B) CCK-8 assays for cell proliferation of HCCLM3-wt and HCCLM3-IL-6(−) cells revealed that the knockout of IL-6 increased the growth inhibition effect induced by 5 and 10 µmol/l sorafenib (***P<0.001 and *P<0.05, respectively). (C and D) Flow cytometric apoptosis assay of HCCLM3-wt and HCCLM3-IL-6(−) cells revealed that the knockout IL-6 increased the apoptosis induced by 5 and 10 µmol/l sorafenib (*P<0.05 and **P<0.01, respectively). (E) Western blot analysis revealed that the level of anti-apoptotic marker Bcl-2 and cell cycle markers cyclin D1 and CDK2 were lower in HCCLM3-IL-6(−) than HCCLM3-wt cells, whereas pro-apoptotic markers cleaved caspase-3 and cleaved PARP were higher in HCCLM3-IL-6(−) than in HCCLM3-wt cells following the administration of 5 and 10 µmol/l sorafenib (all ****P<0.0001). HCC, hepatocellular carcinoma; wt, wild-type; Bcl-2, B-cell lymphoma-2; IL-6, interleukin-6.

Figure 7.

Figure 7.

IL-6 induces liver cancer EMT through JAK/STAT3/Snail pathway hyperactivation. (A and B) Exogenous IL-6 hyperactivation of p-JAK2, p-STAT3 and increased Snail expression in HepG2-wt cells, whereas AG490 blocked the effect induced by IL-6 (all ****P<0.0001). (C and D) IL-6 knockout decreased p-JAK2, p-STAT3 and Snail expression in HCCLM3-IL-6(−) cells, as compared with HCCLM3-wt cells (all ****P<0.0001). (E) CCK-8 assay revealed that exogenous IL-6 could promote HepG2-wt cell proliferation, while this effect was significantly blocked by AG490 (***P<0.001). IL-6, interleukin-6; EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; wt, wild-type.

Similar articles

Cited by

References

    1. Forner A, Reig M, Bruix J. Hepatocellular carcinoma. Lancet. 2018;391:1301–1314. doi: 10.1016/S0140-6736(18)30010-2. - DOI - PubMed
    1. Bruix J, Sherman M, American Association for the Study of Liver Diseases Management of hepatocellular carcinoma: An update. Hepatology. 2011;53:1020–1022. doi: 10.1002/hep.24199. - DOI - PMC - PubMed
    1. Faber W, Seehofer D, Neuhaus P, Stockmann M, Denecke T, Kalmuk S, Warnick P, Bahra M. Repeated liver resection for recurrent hepatocellular carcinoma. J Gastroenterol Hepatol. 2011;26:1189–1194. doi: 10.1111/j.1440-1746.2011.06721.x. - DOI - PubMed
    1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66:7–30. doi: 10.3322/caac.21332. - DOI - PubMed
    1. Gauthier A, Ho M. Role of sorafenib in the treatment of advanced hepatocellular carcinoma: An update. Hepatol Res. 2013;43:147–154. doi: 10.1111/j.1872-034X.2012.01113.x. - DOI - PMC - PubMed

MeSH terms

Substances

Grants and funding

The present study was supported by the National Science Foundation for Young Scientists of China (grant no. 81101851).

LinkOut - more resources