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. 2019 Feb:40:446-456.
doi: 10.1016/j.ebiom.2018.12.047. Epub 2018 Dec 26.

TNF-α is a potential therapeutic target to overcome sorafenib resistance in hepatocellular carcinoma

Wenliang Tan  1 , Xuan Luo  1 , Wenda Li  1 , Jinyi Zhong  2 , Jun Cao  1 , Sicong Zhu  3 , Xianqing Chen  1 , Rui Zhou  1 , Changzhen Shang  4 , Yajin Chen  5
Affiliations

TNF-α is a potential therapeutic target to overcome sorafenib resistance in hepatocellular carcinoma

Wenliang Tan et al. EBioMedicine. 2019 Feb.

Erratum in

Abstract

Background: The role of tumor necrosis factor alpha (TNF-α) in targeted therapy for hepatocellular carcinoma (HCC) remains largely unknown. The current study aimed to clarify the mechanistic effects of targeting TNF-α to overcome sorafenib resistance in HCC.

Methods: A correlation of TNF-α expression with the prognosis was analyzed in 62 HCC patients who underwent surgical resection and subsequent received adjuvant sorafenib treatment. The relation of TNF-α expression and sorafenib sensitivity was determined in different HCC cell lines. The combined therapeutic effects of sorafenib and ulinastatin, which could inhibit TNF-α expression, on HCC were examined in vitro and in vivo.

Findings: High TNF-α expression was correlated with poor outcomes in HCC patients who received adjuvant sorafenib after surgery. In vitro experiments showed that TNF-α promotes HCC cell resistant to sorafenib through inducing epithelial-mesenchymal transition (EMT). Notably, the current study revealed that sorafenib has no significant influence on the expression and secretion of TNF-α, and sorafenib had limited effectiveness on reversing EMT in HCC cells with high TNF-α expression. Inhibiting the expression of TNF-α with ulinastatin significantly enhanced the anti-tumor effect of sorafenib on HCC cells with high expression of TNF-α in vitro and in vivo.

Interpretation: Our findings indicate that TNF-α may serve as a novel predictor of sorafenib sensitivity in HCC patients. Sorafenib combined with ulinastatin may improve the effectiveness of treatment of HCC in patients with high expression of TNF-α. FUND: This work was supported by grants from the National Natural Science Foundation of China (no.81572398; no.81672419), the Science and Technology Planning Project of Guangdong Province (no. 2017A010105003; no.2015A050502023; no.2016A020216010), and the Natural Science Foundation of Guangdong Province (no.2014A030313061; no. 2013B021800101).

Keywords: Combination treatment; Hepatocellular carcinoma; Sorafenib resistance; TNF-α; Ulinastatin.

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Figures

Fig. 1
Fig. 1
High expression of TNF-α was associated with bad prognosis in HCC patients. (a) Representative IHC images showing different levels of TNF-α expression in HCC patients. (b, c) Kaplan–Meier analysis indicated that HCC patients with lower TNF-α expression have better overall survival and recurrence-free survival (n = 62). (d) Analysis of the protein levels of TNF-α in HCC cells by western blot. (e) ELISA assay for the detection of TNF-α secretion in the cultured supernatants of HepG2 and SK-HEP-1 cells. Scale bars: 400 μm (100×); 100 μm (400×). Data represents the mean ± SD of three independent experiments. (*p < .05).
Fig. 2
Fig. 2
TNF-α promoted HCC cell proliferation, invasion, and resistant to sorafenib by inducing EMT. (a) Dose-dependent effects of sorafenib on the viability of HepG2 with or without TNF-α stimulation. (b) Dose-dependent effects of sorafenib on the viability of SK-HEP-1 and SK-HEP-1-sh-TNF-α cells. (c) MTS assays to detect the growth inhibition of sorafenib on HepG2 and SK-HEP-1 cells by the condition of TNF-α stimulation or down-regulation of TNF-α with the shRNA. (d) TNF-α markedly induced EMT in HepG2 cells, while knockdown of TNF-α reversed EMT in SK-HEP-1 cells. (e) The effect of sorafenib on EMT-related markers in HCC cells with or without exogenous TNF-α stimulation. (f) Western blot results showed that sorafenib had no effect on TNF-α expression. Data represents the mean ± SD of three independent experiments. IC50 was calculated by nonlinear regression analysis using Graphpad Prism software. (*p < .05).
Fig. 3
Fig. 3
Ulinastatin inhibited TNF-α expression and enhanced the effect of sorafenib on reversing EMT. (a) Ulinastatin (UTI) suppressed TNF-α expression in a dose-dependent manner. (b) Ulinastatin up-regulated the expression of E-cadherin, and down-regulated the expression of vimentin and snail when the concentration was >1600 U/mL. (c) The effect of sorafenib, ulinastatin on TNF-α secretion in HepG2 and SK-HEP-1 cells as measured by ELISA assay. Lenalidmide (an inhibitor of TNF-α secretion) used as the positive control. (d) The effect of sorafenib on EMT-related markers in HCC cells with or without ulinastatin (1600 U/mL) in HepG2 and SK-HEP-1 cells. Data represents the mean ± SD of three independent experiments. (*p < .05).
Fig. 4
Fig. 4
Ulinastatin enhanced the anti-tumor effect of sorafenib in SK-HEP-1 cells. (a) Colony formation assay showed the anti-proliferation effect of combined sorafenib and ulinastatin in SK-HEP-1 cells. (b) Co-treatment of sorafenib and ulinastatin more strongly induced apoptosis in SK-HEP-1 cells. (c) Western blot assay demonstrated that sorafenib or ulinastatin alone could, to some extent, down-regulate the expression of PCNA (proliferating cell nuclear antigen, a protein to reflect cell proliferation ability) and Bcl-2 (an anti-apoptotic protein), and up-regulate the expression of Bax (a pro-apoptsis protein), while co-treatment of these two drugs displayed better effect for these changes. (d) Representative images showed that the effect of sorafenib, ulinastatin and combined therapy of sorafenib and ulinastatin on SK-HEP-1 cells mobility, and the statistical data of the wound-healing assay. (E) Transwell migration and invasion assays showed that ulinastatin and sorafenib synergistically inhibited cell mobility of SK-HEP-1 cells. (Data represents the mean ± SD of three independent experiments. (*p < .05). (Scale bars in 4a: 4 mm; scale bars in 4d-e: 100 μm).
Fig. 5
Fig. 5
The anti-tumor effect of combined sorafenib and ulinastatin against HCC in vivo. (a) The mice were treated with intragastric administration of vehicle, sorafenib (30 mg/kg/d), or sorafenib and ulinastatin (20,000 U, twice a week, intraperitoneal injection). The photograph shows the dissected tumors from each group. (b) Tumor volumes were measured every 3 days, and tumor growth curves were created for each group. (c) The weight of dissected tumors from each group showed that sorafenib mildly suppressed proliferation of subcutaneous tumors, while combined ulinastatin and sorafenib had a better anti-tumor effect. (d, e) SK-HEP-1 cells (1 × 106/0.2 mL) were injected into the tail vein of the mice to imitate tumor metastasis. Representative hematoxylin and eosin (H&E) staining to assess pulmonary (d) and liver (e) metastasis at 4 weeks. The average number of foci in each group is presented as the mean ± SD. (*p < .05). (Scale bars: 500 μm (40×); 100 μm (200×).
Fig. 6
Fig. 6
Ulinastatin enhanced the anti-tumor effect of sorafenib by suppressing the NF-κB signaling pathway. (a, b) Immunofluorescence staining to show the expression and nuclear translation of P65 in HCC cells. Representative images show that TNF-α markedly induced P65 nuclear translocation, while ulinastatin strongly prevented P65 nuclear translocation. (scale bars: 25 μm). (c) TNF-α was used as a positive control to activate the NF-κB signaling pathway, and BAY 11–7082, an inhibitor specifically inhibit P65 translating to the nuclear, was used as a control for suppressing the NF-κB signaling pathway. Western blot results showed that ulinastatin inhibited the phosphorylation of IKK-β, IκB, and P65, while sorafenib almost had no impact on the phosphorylation of IKK-β, IκB, and P65 in HCC cells. The effect of co-treatment with the two drugs was superior to that of either drug alone. (d) HCC cells were pretreated with or without TNF-α stimulation, and then the cells were treated with sorafenib, BAY 11–7082 or combined sorafenib with BAY 11–7082, MTS assay to detect the growth inhibition for each group. (e) Transwell migration assay to show the combined therapy of sorafenib and BAY 11–7082 on cell mobility with or without TNF-α stimulation. (Scale bars: 100 μm). (Data represents the mean ± SD of three independent experiments. (*p < .05).
Fig. 7
Fig. 7
The combination of sorafenib and ulinastatin suppressed the NF-κB signaling pathway. (a, b) Tumor sections from the subcutaneous tumor in the SK-HEP-1 cell models were stained for TNF-α, P65, vimentin, and Ki67. The results showed that sorafenib alone suppressed Ki67, but had limited effect on TNF-α, vimentin expression, and P65 nuclear translocation, while co-treatment of sorafenib and ulinastatin significantly suppressed the expression of TNF-α, vimentin, and Ki67, and inhibited nuclear translocation of P65. (c) Ulinastatin reverses sorafenib resistance through inhibiting the TNF-α/ NF-κB/EMT signaling pathway in HCC cells. (Data represents the mean ± SD of the IHC score of six independent animals for each group. (*p < .05). (Scale bars in 7a: 100 μm; inserts: 40 μm)
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