Sophoridine suppresses lenvatinib-resistant hepatocellular carcinoma growth by inhibiting RAS/MEK/ERK axis via decreasing VEGFR2 expression

Abstract

Hepatocellular carcinoma (HCC) is one of the most lethal cancer types with insufficient approved therapies, among which lenvatinib is a newly approved multi-targeted tyrosine kinase inhibitor for frontline advanced HCC treatment. However, resistance to lenvatinib has been reported in HCC treatment recently, which limits the clinical benefits of lenvatinib. This study aims to investigate the underlying mechanism of lenvatinib resistance and explore the potential drug to improve the treatment for lenvatinib-resistant (LR) HCC. Here, we developed two human LR HCC cell lines by culturing with long-term exposure to lenvatinib. Results showed that the vascular endothelial growth factor receptors (VEGFR)2 expression and its downstream RAS/MEK/ERK signalling were obviously up-regulated in LR HCC cells, whereas the expression of VEGFR1, VEGFR3, FGFR1-4 and PDGFRα/β showed no difference. Furthermore, ETS-1 was identified to be responsible for VEGFR2 mediated lenvatinib resistance. The cell models were further used to explore the potential strategies for restoration of sensitivity of lenvatinib. Sophoridine, an alkaloid extraction, inhibited the proliferation, colony formation, cell migration and increased apoptosis of LR HCC cells. In vivo and in vitro results showed Sophoridine could further sensitize the therapeutic of lenvatinib against LR HCC. Mechanism studies revealed that Sophoridine decreased ETS-1 expression to down-regulate VEGFR2 expression along with downstream RAS/MEK/ERK axis in LR HCC cells. Hence, our study revealed that up-regulated VEGFR2 expression could be a predicator of the resistance of lenvatinib treatment against HCC and provided a potential candidate to restore the sensitivity of lenvatinib for HCC treatment.

Keywords: VEGFR2; hepatocellular carcinoma; lenvatinib resistance; sophoridine.

Figure 1

Development of lenvatinib‐resistant cell lines. A, The cell viability was measured by CCK‐8 assay at different time intervals (24, 48, 72 and 96 h) when cells were cultured with 5 μmol/L lenvatinib. The proliferation of lenvatinib‐resistant (LR) HepG2 or Huh7 cells was greater than parental HepG2 or Huh7 cells (WT). control: parental HepG2 or Huh7 cells without lenvatinib treatment; WT: parental HepG2 or Huh7 cells with lenvatinib treatment; LR: lenvatinib‐resistant HepG2 or Huh7 cells with lenvatinib treatment. B, Table with IC₅₀ values of lenvatinib against the two developed resistant cell lines and their corresponding parental cells. C, Cells were exposed to 5 μmol/L lenvatinib in clonogenicity assay. Represent clone images of different indicated cells were shown. D, Quantification of the number of clones in different cells after treatment with 5 μmol/L lenvatinib. LR‐HepG2 or LR‐Huh7 cells formed more clones than their parental cells. ns, P value > 0.05; ***, P value < 0.001

Figure 2

Upregulated VEGFR2 expression mediates lenvatinib resistance by activating RAS/MEK/ERK signalling. A, The mRNA expression of VEGFR2 in HepG2‐WT/LR and Huh7‐WT/LR cells. VEGFR2 mRNA expression was up‐regulated in HepG2‐LR and Huh7‐LR cells. B, The protein expression of VEGFR2 in HepG2‐WT/LR and Huh7‐WT/LR cells. C, Western blotting results showed the downstream target RAS/MEK/ERK axis of VEGFR2 were obviously up‐regulated in HepG2‐LR and Huh7‐LR cells compared with their parental cells. ***, P value < 0.001

Figure 3

ETS‐1 was responsible for VEGFR2 mediated lenvatinib resistance. A, The mRNA expression of ETS‐1 in HepG2‐WT/LR and Huh7‐WT/LR cells. ETS‐1 mRNA expression was increased in HepG2‐LR and Huh7‐LR cells. B, The protein expression of ETS‐1 in HepG2‐WT/LR and Huh7‐WT/LR cells. C and D, HepG2‐LR and Huh7‐LR cells were transfected with control siRNA (NC) or ETS‐1 siRNA. The knock down efficiency of ETS‐1 siRNA was detected. The mRNA and protein expression of ETS‐1 were quantified by qPCR and Western blotting. E, The mRNA expression of VEGFR2 after knocking down ETS‐1 expression in HepG2‐LR and Huh7‐LR cells. F, The indicated cells were treated with 5 μmol/L lenvatinib for 48 h, and then, cell viability was detected by CCK‐8 assay. ETS‐1 knock down rescued the efficacy of lenvatinib against lenvatinib‐resistant HCC cells. Control: parental HepG2 or Huh7 cells; WT: parental HepG2 or Huh7 cells with lenvatinib treatment; WT + siRNA: parental HepG2 or Huh7 cells were firstly transfected with ETS‐1 siRNA and then treated with lenvatinib; LR: lenvatinib‐resistant HepG2 or Huh7 cells with lenvatinib treatment; LR + siRNA: lenvatinib‐resistant HepG2 or Huh7 cells were firstly transfected with ETS‐1 siRNA and then treated with lenvatinib. ns, P value > 0.05; *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001

Figure 4

Sophoridine inhibited the proliferation, colony formation and increased apoptosis of lenvatinib‐resistant HCC cells. A, Lenvatinib‐resistant (LR) HepG2 or Huh7 cells were treated with different doses (20, 40 and 80 μmol/L) of Sophoridine (SPO) for 72 h. The viability of HepG2‐LR and Huh7‐LR cells was determined by CCK‐8 assay. Sophoridine treatment suppressed the growth of HepG2‐LR and Huh7‐LR cells. B, The HepG2‐LR and Huh7‐LR cells were treated with 20, 40 or 80 μmol/L Sophoridine in colony formation assay. The colonies formation of HepG2‐LR and Huh7‐LR cells were inhibited after Sophoridine treatment. C, Represent clone images were shown in different groups after indicated treatments. D, The apoptosis of HepG2‐LR and Huh7‐LR cells were detected by the Annexin‐V/PI staining assay through flow cytometry after 20, 40 or 80 μmol/L Sophoridine treatment for 24 h. Sophoridine induced the apoptosis of HepG2‐LR and Huh7‐LR cells. E, The representative gating images of Sophoridine on the HepG2‐LR or Huh7‐LR cells apoptosis were shown. **, P value < 0.01; ***, P value < 0.001

Figure 5

Sophoridine suppressed the migration of lenvatinib‐resistant HepG2 and Huh7 cells. A, B, Lenvatinib‐resistant (LR) HepG2 or Huh7 cells were previously treated with 20, 40 or 80 μmol/L of Sophoridine for 24 h. Representative images of the migration ability of HepG2‐LR and Huh7‐LR cells were shown. C, The numbers of migrated HepG2‐LR and Huh7‐LR cells were quantified. *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001

Figure 6

Sophoridine sensitized the anti‐tumour effect of lenvatinib against lenvatinib‐resistant HCC cell lines in vitro and in vivo. A, Lenvatinib‐resistant (LR) HepG2 or Huh7 cells were treated with 20 μmol/L Sophoridine combined with 5 μmol/L lenvatinib for 72 h. The combination treatment showed the most effective influence on the growth of HepG2‐LR and Huh7‐LR cells. B, The HepG2‐LR and Huh7‐LR cells were treated with 20 μmol/L Sophoridine combined with 5 μmol/L lenvatinib in colony formation assay. Colonies formation of HepG2‐LR and Huh7‐LR cells were inhibited after combination treatment. C, Representative images of the colony formation ability of HepG2‐LR and Huh7‐LR cells were shown after indicated treatments. D, BALB/c nude mice were subcutaneously burdened with HepG2‐LR cells. Mice were divided into different treatment groups after 7 days (n = 6). Mice were treated with Sophoridine (50 mg/kg, daily, intraperitoneally), lenvatinib (30 mg/kg, daily, intragastrically) or Sophoridine combined with lenvatinib. Tumour growth change curves were explored after indicated treatment. E, Tumour weight was detected after mice killing. F, The relative tumour volume (RTV) ratio was calculated according to the formula RTV = tumour volume day 16‐day 0/tumour volume day 0. G, Mouse bodyweight changes were observed during the treatments. H, The representative photograph of tumours was shown in different groups. I, Tumour slices of indicated groups were stained with Ki67, CD31, VEGFR2 and p‐ERK. The representative immunohistochemistry images of Ki67, CD31, VEGFR2 and p‐ERK from different groups were displayed. ns, P value > 0.05; **, P value < 0.01; ***, P value < 0.001

Figure 7

Sophoridine down‐regulated VEGFR2 expression along with downstream RAS/MEK/ERK axis in lenvatinib‐resistant HCC cells. A, Lenvatinib‐resistant (LR) HepG2 or Huh7 cells were treated with different doses of Sophoridine for 24 h. Sophoridine treatment down‐regulated ETS‐1 and VEGFR2 mRNA expression in HepG2‐LR and Huh7‐LR cells. B, The protein expression of ETS‐1 and VEGFR2 was detected by Western blotting in HepG2‐LR and Huh7‐LR cells after Sophoridine treatment. C, The downstream RAS/MEK/ERK axis was significantly inhibited in Sophoridine‐treated HepG2‐LR and Huh7‐LR cells. D, The indicated cells were treated with 40 μmol/L Sophoridine for 72 h, and then, the cell viability was detected by CCK‐8 assay. Control: HepG2‐LR or Huh7‐LR cells; SPO: HepG2‐LR or Huh7‐LR cells with Sophoridine treatment; siRNA: HepG2‐LR or Huh7‐LR cells were transfected with ETS‐1 siRNA; SPO + siRNA: HepG2‐LR or Huh7‐LR cells were firstly transfected with ETS‐1 siRNA and then treated with Sophoridine. ns, P value > 0.05; *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001