FBP1 induced by β-elemene enhances the sensitivity of gefitinib in lung cancer

Abstract

Background: β-elemene is known to play a critical role in tumorigenesis as well as tyrosine kinase inhibitor (TKI) resistance in lung cancer. However, the biological function and molecular mechanism remain largely unknown.

Methods: In this study, the common genes involved in gefitinib resistance and β-elemene were identified using bioinformatic analysis. The expression of FBP1 was examined by qRT-PCR and Western blot analysis. Cell proliferation, flow cytometry, clone formation and IC50 assays were performed to assess the effects of β-elemene and FBP1. Western blot analysis was used to evaluate apoptosis-related gene expression. Finally, in vivo experiments were conducted to assess the crucial role of FBP1 in gefitinib-resistant HCC827/GR cells in nude mice.

Results: Screening analysis demonstrated that fructose-1,6-bisphosphatase (FBP1) was induced by β-elemene and downregulated in gefitinib-resistant lung cells. Functionally, overexpression of FBP1 inhibited proliferation and gefitinib resistance and promoted apoptosis of PC9/GR and HCC827/GR cells in vitro. Mechanistically, FBP1 impeded the nuclear translocation of p-STAT3. The FBP1/STAT3 axis was required for FBP1-mediated apoptosis-related gene expression. In vivo experiments further confirmed the enhanced effects of FBP1 on lung cancer cell sensitivity to gefitinib.

Conclusion: Our research indicated that β-elemene suppressed proliferation and enhanced sensitivity to gefitinib by inducing apoptosis through the FBP1/STAT3 axis in gefitinib-resistant lung cancer cells.

Keywords: FBP1; STAT3; gefitinib; lung cancer; β-elemene.

FIGURE 1

Downregulation of FBP1 in gefitinib‐resistant lung cells. (a) Simplified β‐elemene‐key target network. (b) Heatmap depicting the differentially expressed genes between parental HCC4006 cells and gefitinib‐resistant HCC4006 cells based on GSE123066 datasheets. (c) Venn diagram of β‐elemene targets and the downregulated genes in gefitinib‐resistant HCC4006 cells from GSE123066 datasheets. (d) The mRNA and protein levels of FBP1 in PC9, PC9/GR, HCC827, and HCC827/GR cells. (e). The mRNA and protein levels of FBP1 in PC9/GR and HCC827/GR cells with or without 10 μg/ml β‐elemene treatment. (f) Representative images of the dose‐dependent increase in FBP1 expression after β‐elemene treatment. ** p < 0.01

FIGURE 2

Overexpression of FBP1 inhibited cell growth and induced apoptosis in gefitinib‐resistant lung cells. (a) The expression of FBP1 was examined in PC9/GR and HCC827/GR cells after FBP1 overexpression by Western blot. (b) Growth curve of PC9/GR and HCC827/GR cells after FBP1 overexpression by CCK‐8 assay. (c) Flow cytometry was performed to assess the apoptosis level of PC9/GR and HCC827/GR cells after FBP1 overexpression and 10 μM gefitinib treatment. (d) The IC50 values of gefitinib in PC9/GR and HCC827/GR cells were analyzed using GraphPad. (e) A colony forming experiment was conducted in PC9/GR and HCC827/GR cells after FBP1 overexpression. ** p < 0.01

FIGURE 3

FBP1‐mediated sensitivity to gefitinib in PC9/GR and HCC827/GR cells. PC9/GR and HCC827/GR were transduced with SCR or shFBP1 (shRNA targeting FBP1) and then treated with 10 μg/ml β‐elemene for 24 h for subsequent experiments. (a) The protein level of FBP1 was measured in PC9/GR and HCC827/GR cells. (b) Growth curve, (c) apoptosis level, (d) colony formation, and (e) IC50 values of gefitinib in PC9/GR and HCC827/GR cells as indicated treatment. **p < 0.01, compared with PBS; # p < 0.05, compared with SCR

FIGURE 4

STAT3 was required for the FBP1‐mediated apoptosis pathway. (a) The protein expression of STAT3 and p‐STAT3 (Tyr705) in PC9/GR cells after FBP1 overexpression in the nucleus and cytoplasm. (b) The protein expression of STAT3 and p‐STAT3 (Tyr705) in HCC827/GR cells after FBP1 overexpression in the nucleus and cytoplasm. (c) Western blot analysis was used to evaluate the expression of BCL‐2, Bax, and cleaved caspase‐3 in PC9/GR and HCC827/GR cells after FBP1 overexpression. (d) Western blot analysis was used to evaluate the expression of BCL‐2, Bax, and cleaved caspase‐3 in PC9/GR and HCC827/GR cells after FBP1 silencing. (E and F) the protein expression of STAT3 and p‐STAT3 (Tyr705) in PC9/GR (e) and HCC827/GR (f) cells after FBP1 overexpression with or without colivelin treatment in the nucleus, as well as the expression of BCL‐2, Bax, and cleaved caspase‐3 in the cytoplasm

FIGURE 5

β‐elemene regulates gefitinib resistance through the FBP1/STAT3 axis in PC9/GR and HCC827/GR cells. (a and b) The protein expression of STAT3 and p‐STAT3 (Tyr705) in PC9/GR (a) and HCC827/GR (b) cells after β‐elemene treatment, followed by or without 0.5 μM colivelin treatment in the nucleus. The protein expression of BCL‐2, Bax, and cleaved caspase‐3 in PC9/GR (a) and HCC827/GR (b) cells after β‐elemene treatment, followed by or without colivelin treatment in the cytoplasm. (c) Growth curve, (d) apoptosis level, (e) colony formation, and (f) IC50 values of gefitinib in PC9/GR and HCC827/GR cells as indicated treatment. **p < 0.01, compared with PBS; # p < 0.05, compared with DMSO

FIGURE 6

The overexpression of FBP1 inhibited tumor growth in vivo. (a) The xenograft tumors from nude mice with HCC827/GR transduced with vector or FBP1. (b) The growth curve of nude mice in the vector group and FBP1 group. (c) Tumor weight of the xenograft tumors from nude mice with HCC827/GR cells transduced with vector or FBP1. **p < 0.01

FIGURE 7

The working model describes the mechanism of FBP1. The expression of FBP1 was increased in gefitinib‐resistant lung cancer cells when stimulated by β‐elemene. The upregulation of FBP1 bound to STAT3 and inhibited the phosphorylation of STAT3, resulting in the altered expression of apoptosis‐related genes and leading to gefitinib sensitivity