Qing-Kai-Ling Injection Acts Better Than Shen-Fu Injection in Enhancing the Antitumor Effect of Gefitinib in Resistant Non-Small Cell Lung Cancer Models

Abstract

Patients with EGFR gene mutation often obtain de novo resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) or develop secondary resistance to EGFR-TKIs after taking EGFR-TKI therapy. Traditional Chinese medicine (TCM) with different treatment principles, in combination with EGFR-TKIs, plays an important role in the treatment of cancers including resistant non-small cell lung cancer (NSCLC). However, inappropriate use of TCM herbs may induce resistance to gefitinib. Therefore, it is of a great value to evaluate which TCM treatment principle should be combined with EGFR-TKIs, and which one should be avoided, and find out the potential mechanisms. The lentiviral transfection assay was used for overexpression of PIK3CA mutation gene in PC-9 cells to construct PC-9-PIK3CA-mutation (PC-9-PIK3CA-M) cells. Cell proliferation, apoptosis, and the expression of EGFR/PI3K/AKT and EGFR/RAS/RAF/ERK in PC-9-PIK3CA-M and H1975 cells treated by the typical cooling-heat drug, Qing-kai-ling (QKL) and Tan-re-qing (TRQ), or the typical warming-yang drug, Shen-fu (SF) and gefitinib treatment, were detected by MTT, Annexin V/PI double labeling, and Western blot assays, respectively. Tumor xenograft and immunohistochemistry experiments were carried out to confirm the in vitro findings. PC-9-PIK3CA-M cells were less sensitive to gefitinib, when compared with PC-9 cells. QKL injection and TRQ injection, not SF injection, combined with gefitinib induced significantly increased cell growth inhibition and apoptosis in PC-9-PIK3CA-M and H1975 cells. SF injection antagonized the effect of gefitinib in promoting cancer cell apoptosis. QKL injection and TRQ injection increased the sensitivity of gefitinib by inhibiting the phosphorylation of AKT or ERK in H1975 and PC-9-PIK3CA-M cells. Similar findings were observed in vivo in H1975 xenograft mouse model. QKL and TRQ, with cooling-heat TCM treatment principle, should be combined with gefitinib in the treatment of NSCLC. Furthermore, warming-yang drug SF should be avoided to be used together with EGFR-TKIs.

Figure 1

PC-9-PIK3CA-M cells are less sensitive to gefitinib due to the activated PIK3CA-AKT signal pathway: (a) Western blot assay detecting the protein expression of PIK3CA, AKT, and p-AKT in PC-9 and PC-9-PIK3CA-M cells. (b) MTT assay detecting the cell viability after the treatment with gefitinib in PC-9 and PC-9-PIK3CA-M cells. (c) Western blot assay detecting the protein expression of AKT, p-AKT after the treatment with gefitinib in PC-9 and PC-9-PIK3CA-M cells. Data are presented as the mean ± standard deviation of three independent experiments. ^∗P < 0.05 vs. control group. PC-9-PIK3CA-M, PC-9-PIK3CA-mutation.

Figure 2

QKL injection and TRQ injection, but not SF injection, increased the sensitivity of gefitinib in PC-9-PIK3CA-M and H1975 cells: (a) MTT assay on the cell viability after the treatment with QKL/SF and gefitinib for 48 h and 72 h in PC-9-PIK3CA-M cells. (b) MTT assay detecting the cell viability after the treatment with QKL/SF and gefitinib for 48 h and 72 h in H1975 cells. (c) MTT assay detecting the cell viability after the treatment with TRQ and gefitinib for 48 h in PC-9-PIK3CA-M and H1975 cells. Data are presented as the mean ± standard deviation of three independent experiments. ^∗P < 0.05 vs. gefitinib alone, ^▲P < 0.05 vs. QKL/SF alone. QKL, Qing-kai-ling injection; SF, Shen-fu injection; TRQ, Tan-re-Qing injection.

Figure 3

QKL injection increased, and SF injection antagonized the proapoptotic effect of gefitinib in PC-9-PIK3CA-M and H1975 cells: (a) apoptosis assay to assess the combination effect of gefitinib and QKL/SF for 48 h in PC-9-PIK3CA-M cells. (b) Apoptosis assay to assess the combination effect of gefitinib and QKL/SF for 48 h in H1975 cells. (c) The statistics of apoptotic rate induced by gefitinib and QKL in PC-9-PIK3CA-M and H1975 cells. (d) The statistics of apoptotic rate induced by gefitinib and SF in PC-9-PIK3CA-M and H1975 cells. Data are presented as the mean ± standard deviation of three independent experiments. ^∗P < 0.05 vs. control group, ^▲P < 0.05 vs. gefitinib group, and ^■P < 0.05 vs. QKL/SF group. QKL, Qing-kai-ling injection; SF, Shen-fu injection.

Figure 4

QKL/TRQ injection, not SF injection, enhanced the inhibition of the downstream signaling pathways of EGFR induced by gefitinib in vitro. (a) Western blot showing the expression of proteins related to the combination effect of gefitinib and QKL/SF in PC-9-PIK3CA-M and H1975 cell lines. (b) The effects of gefitinib and QKL/SF on EGFR, p-EGFR, AKT, p-AKT, ERK, and p-ERK protein expression in PC-9-PIK3CA-M cells and H1975 cells. (c) Western blot showing the expression of proteins related to the combination effect of gefitinib and TRQ in PC-9-PIK3CA-M and H1975 cell lines. (e) The effects of gefitinib and TRQ on EGFR, p-EGFR, AKT, p-AKT, ERK, and p-ERK protein expression in PC-9-PIK3CA and H1975 cells. Data are presented as the mean ± standard deviation of three independent experiments. ^∗P < 0.05 vs. control. ^▲P < 0.05 vs. gefitinib alone. QKL, Qing-kai-ling injection; SF, Shen-fu injection; TRQ, Tan-re-qing injection.

Figure 5

QKL oral solution, not SF decoction, and gefitinib synergistically inhibited the tumor growth via regulating p-AKT in vivo. (a) The tumor growth curve showing the anticancer effect of gefitinib and QKL/SF in H1975 xenograft transplanted nude mice. (b) The tumor weight showing the anticancer effect of gefitinib and QKL/SF in H1975 xenograft transplanted nude mice at day 19. (c) The statistics of EGFR, p-EGFR, AKT, p-AKT, ERK, and p-ERK protein expression in H1975 xenograft transplanted nude mice. (d) Immunohistochemical staining showing the effects of gefitinib and QKL/SF on the expression of EGFR, p-EGFR, AKT, p-AKT, ERK, and p-ERK protein (×400 magnifications). ^∗P < 0.05 vs. control group. ^▲P < 0.05 vs. gefitinib group. QKL, Qing-kai-ling oral solution; SF, Shen-fu decoction.