Active fractions of golden-flowered tea (Camellia nitidissima Chi) inhibit epidermal growth factor receptor mutated non-small cell lung cancer via multiple pathways and targets in vitro and in vivo

Abstract

As a medicine-food homology (MFH) plant, golden-flowered tea (Camellia nitidissima Chi, CNC) has many different pharmacologic activities and is known as "the queen of the tea family" and "the Panda of the Plant world". Several studies have revealed the pharmacologic effects of CNC crude extract, including anti-tumor, anti-oxidative and hepatoprotective activity. However, there are few studies on the anti-tumor active fractions and components of CNC, yet the underlying mechanism has not been investigated. Thus, we sought to verify the anti-non-small cell lung cancer (NSCLC) effects of four active fractions of CNC. Firstly, we determined the pharmacodynamic material basis of the four active fractions of CNC (Camellia. leave. saponins, Camellia. leave. polyphenols, Camellia. flower. saponins, Camellia. flower. polyphenols) by UPLC-Q-TOF-MS/MS and confirmed the differences in their specific compound contents. Then, MTT, colony formation assay and EdU incorporation assay confirmed that all fractions of CNC exhibit significant inhibitory on NSCLC, especially the Camellia. leave. saponins (CLS) fraction on EGFR mutated NSCLC cell lines. Moreover, transcriptome analysis revealed that the inhibition of NSCLC cell growth by CLS may be via three pathways, including "Cytokine-cytokine receptor interaction," "PI3K-Akt signaling pathway" and "MAPK signaling pathway." Subsequently, quantitative real-time PCR (RT-qPCR) and Western blot (WB) revealed TGFB2, INHBB, PIK3R3, ITGB8, TrkB and CACNA1D as the critical targets for the anti-tumor effects of CLS in vitro. Finally, the xenograft models confirmed that CLS treatment effectively suppressed tumor growth, and the key targets were also verified in vivo. These observations suggest that golden-flowered tea could be developed as a functional tea drink with anti-cancer ability, providing an essential molecular mechanism foundation for MFH medicine treating NSCLC.

Keywords: Camellia nitidissima Chi (CNC); epidermal growth factor receptor (EGFR); golden-flowered tea; natural product; non-small cell lung cancer (NSCLC).

FIGURE 1

Experimental design ideas.

FIGURE 2

Chemical composition analysis of CLS, CLP, CFS, and CFP by using UPLC-QTOF-MS. (A) Total Ion Chromatography (TIC) in the positive and negative ion mode of CLS by UPLC-QTOF-MS. (B) Total Ion Chromatography (TIC) in the positive and negative ion mode of CLP by UPLC-QTOF-MS. (C) Total Ion Chromatography (TIC) in the positive and negative ion mode of CFS by UPLC-QTOF-MS. (D) Total Ion Chromatography (TIC) in the positive and negative ion mode of CFP by UPLC-QTOF-MS/MS.

FIGURE 3

Inhibition of proliferation and clonogenic ability of NSCLC cell lines by four active fractions of CNC. (A) Inhibitory effects of different concentrations of CLP, CLS, CFP, and CFS (6.25, 12.5, 25, 50 μg⋅mL^–1) on NCI-H1975, HCC827 and A549 cells, respectively. (B) IC50 of different concentrations of CLP, CLS, CFP, and CFS (6.25, 8, 10, 12.5, 15, 20, 25, and 50 μg⋅mL^–1) on NCI-H1975, HCC827, and A549 cells, respectively. (C) Inhibitory effects of different concentrations of CLS, CLP, CFS, and CFP (1.5625, 3.125, 6.25 μg⋅mL^–1) on colony formation of NCI-H1975 cells, respectively. *Indicates p < 0.05, **indicates p < 0.01 and *** indicates p < 0.001 relative to the control by ANOVA. The data are presented as the mean ± standard deviation (n = 3).

FIGURE 4

Inhibition of proliferation rate of NCI-H1975 cells by four active fractions of CNC. (A–D) Representative field of view of immunofluorescence assay with anti-EdU antibody is shown, where in NCI-H1975 cells, the signal of incorporating with EdU was green and the nucleus was stained blue by Hoechst. Histograms showed the mean values of cell proliferation rates after administration of different concentrations of CLS, CLP, CFS, and CFP (6.25, 12.5, 25 μg⋅mL^–1). *Indicates p < 0.05, **indicates p < 0.01 and ***indicates p < 0.001 relative to the control by ANOVA. The data are presented as the mean ± standard deviation (n = 3).

FIGURE 5

CLS induced programmed NSCLC death through pyroptosis. (A) TdT-mediated dUTP Nick-End Labeling (TUNEL) positive rate of NCI-H1975 cells after administration of different concentrations of CLS (6.25, 12.5, 25 μg⋅mL^–1). (B) Reactive oxygen species (ROS) positive rate of NCI-H1975 cells after administration of different concentrations of CLS (6.25, 12.5, 25 μg⋅mL^–1). (C) Annexin V-FITC/propidine iodide (PI) staining of NCI-H1975 cells after 48 h was performed to identify early/late apoptosis, and the data were analyzed via flow cytometry. (D) SEM was used to detect the morphological changes of NCI-H1975 cells. (E,F) Relative uperoxide dismutase (SOD) activity and relative lactate dehydrogenase (LDH) activity of NCI-H1975 cells after administration of different concentrations of CLS (6.25, 12.5, 25 μg⋅mL^–1). (G) Relative IL-1β activity and relative LDH activity of NCI-H1975 cells after administration of different concentrations of CLS (6.25, 12.5, 25 μg⋅mL^–1). *Indicates p < 0.05, **indicates p < 0.01 and ***indicates p < 0.001 relative to the control by ANOVA. The data are presented as the mean ± standard deviation (n = 3).

FIGURE 6

Transcriptome analysis of NCI-H1975 cells between the control group and CLS-treated group. (A) Volcano plot with 1,077 DEGs up-regulated and 1,008 DEGs down-regulated. (B) GO enrichment analysis of up-regulated and down-regulated DEGs. (C) KEGG enrichment analysis of up-regulated and down-regulated DEGs. (D) The expression heatmap of 22 DEGs related to Cytokine-cytokine receptor interaction and Rheumatoid arthritis signaling pathway in each sequencing sample. (E) The expression heatmap of 23 DEGs related to PIK-Akt signaling pathway in each sequencing sample. (F) The expression heatmap of 16 DEGs related to MAPK signaling pathway in each sequencing sample. (H,I) Interaction relationships of DEGs on four signaling pathways, including TGF-β signaling pathway, TNF signaling pathway, PIK-Akt signaling pathway and MAPK signaling pathway.

FIGURE 7

Comparative analysis of quantitative real-time PCR (RT-qPCR) data and Western Blot (WB) data. (A) Relative mRNA levels of nine genes including TGFB2, INHBB, TNFRSF10C, PIK3R3, ITGB8, EIF4E1B, NTRK, CACN1D, MEF2C. (B) Protein levels of INHBB, PIK3R3, TGFB2, ITGB8, TrkB, CACNA1D detected in CLS-treated NCI-H1975 cells were detected by Western blot with quantification of each protein, n = 3 biologically independent embryos. *Indicates p < 0.05, **indicates p < 0.01 and ***indicates p < 0.001 relative to the control by ANOVA. The data are presented as the mean ± standard deviation (n = 3).

FIGURE 8

CLS suppressed tumor growth in nude mice. (A,B) Signs and weight changes in nude mice, n = 6 biologically independent embryos. (C,D) Variations in relative tumor volume (RTV) and tumor size in nude mice, n = 6 biologically independent embryos. (E) Tumor weight in nude mice, n = 6 biologically independent embryos. (F,G) Morphological observation of tumor by H&E staining and Ki67 detected by DAB yellow staining. (H,I) Protein levels of INHBB, PIK3R3, TGFB2, ITGB8, TrkB, CACNA1D detected in CLS-treated tumors were detected by Western blot with quantification of each protein, n = 3 biologically independent embryos. *p < 0.05, **p < 0.01, and ***p < 0.001 relative to the control by ANOVA.