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. 2018 Jan;15(1):109-114.
doi: 10.3892/ol.2017.7293. Epub 2017 Oct 30.

Sulforaphane reverses gefitinib tolerance in human lung cancer cells via modulation of sonic hedgehog signaling

Fanping Wang  1   2 , Wenjun Wang  3 , Junpeng Li  4 , Jingjing Zhang  1 , Xia Wang  1 , Mingyong Wang  1
Affiliations

Sulforaphane reverses gefitinib tolerance in human lung cancer cells via modulation of sonic hedgehog signaling

Fanping Wang et al. Oncol Lett. 2018 Jan.

Abstract

Gefitinib is a targeted anticancer drug that was developed as an effective clinical therapy for lung cancer. Numerous patients develop gefitinib resistance in response to treatment. Sulforaphane (SFN) is present in cruciferous vegetables, and has been demonstrated to inhibit the malignant growth of various types of cancer cells. To investigate the role of SFN in gefitinib resistance, a gefitinib-tolerant PC9 (PC9GT) cell model was established by continually exposing PC9 cells to gefitinib. Cell viability was measured using a cell proliferation assay. Components of the sonic hedgehog (SHH) signaling pathway and markers of lung cancer stem cells were detected via western blotting. SFN markedly inhibited the proliferation of PC9GT and PC9 cells in a dose-dependent manner; combination SFN/gefitinib treatment also markedly decreased PC9GT cell proliferation, compared with SFN or gefitinib administered alone (P<0.05). Western blot analysis revealed that the expression of SHH, Smoothened (SMO), zinc finger protein GLI1 (GLI1), GLI2, CD133 and CD44 were upregulated in PC9GT cells, as compared with in PC9 cells. Furthermore, SFN markedly inhibited the expression of SHH, SMO and GLI1 in PC9GT and PC9 cells in a dose dependent manner, and SFN combined with gefitinib markedly inhibited the expression of SHH, SMO, GLI1, CD133 and CD44 in PC9GT cells when compared with SFN or gefitinib monotherapy. The results of the present study demonstrated that SFN inhibits the proliferation of gefitinib-tolerant lung cancer cells via modulation of the SHH signaling pathway. Therefore, combined SFN and gefitinib therapy may be an effective approach for the treatment of lung cancer.

Keywords: gefitinib tolerance; lung cancer; sonic hedgehog signaling pathway; sulforaphane.

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Figures

Figure 1.
Figure 1.
Sensitivity to gefitinib was determined using a cell proliferation assay. (A) PC9GT and PC9 cells were separated into two groups. One group was treated with gefitinib and the other remained untreated. (B) The cell counting assay demonstrated that PC9GT cells continued to proliferate, unlike the PC9 cells (*P<0.05). (C) The MTT assay showed that the PC9GT cells were able to continue proliferating when exposed to 2 µmol/l gefitinib, contrary to PC9 cells (*P<0.05, compared with PC9 cells). SFN, sulforaphane; PC9GT, gefitinib-tolerant PC9 cells.
Figure 2.
Figure 2.
The expression of key SHH and the marker of LSCs in lung cancer cells was analyzed via western blotting. (A) The expression of SHH, SMO, GLI1 and GLI2 was high in PC9GT cells, as compared with in PC9 cells. (B) The expression of CD133 and CD44 was upregulated in PC9GT cells, compared with in PC9 cells. (C) SFN markedly inhibited the expression of SHH, SMO and GLI1 in PC9GT and PC9 cells, in a dose-dependent manner. SFN, sulforaphane; PC9GT, gefitinib-tolerant PC9 cells; EGFR, epidermal growth factor receptor; SHH, sonic hedgehog; SMO, smoothened; GLI1/2, zinc finger protein GLI1/2.
Figure 3.
Figure 3.
PC9 and PC9GT cell proliferation was affected by SFN treatment. (A) SFN markedly inhibited the proliferation of PC9GT and PC9 in a dose-dependent manner. (B) The tumorsphere volumes were not significantly different between the PC9GT and PC9 cells. (C) PC9GT cells were treated with gefitinib, SFN and combined therapy with SFN and gefitinib. Combined treatment markedly decreased PC9GT cell density, as observed by microscopy. SFN, sulforaphane; PC9GT, gefitinib-tolerant PC9 cells.
Figure 4.
Figure 4.
The effect of combined SFN and gefitinib therapy on PC9GT cells was determined using a cell proliferation assay and western blotting. (A) Combined treatment markedly decreased PC9GT cell proliferation, when compared with SFN or gefitinib monotherapy, at 48 and 72 h (*P<0.05, compared with SFN, gefitinib and the control). (B) SFN combined with gefitinib markedly inhibited the expression of SHH, SMO, GLI1, CD133 and CD44 in PC9GT cells, as compared with SFN or gefitinib administered alone. SFN, sulforaphane; PC9GT, gefitinib-tolerant PC9 cells; EGFR, epidermal growth factor receptor; SHH, sonic hedgehog; SMO, Smoothened; GLI1, zinc finger protein GLI1.
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