Shenqi Fuzheng injection restores the sensitivity to gefitinib in non-small cell lung cancer by inhibiting the IL-22/STAT3/AKT pathway

Figures & data

Table 1. Primers for quantitative PCR analysis.

Gene	Primer sequences (5’-3’)
GAPDH	F: GTCTCCTCTGACTTCAACAGCG
	R: ACCAGGCGCCCAATACGACCAA
IL-22	F: CCTCAATCTGATAGGTTCCA
	R: TCATCACCTTCAATATGACATG
IL-22RA1	F: GCTGACCATCTTGACTGT
	R: AGTTGCTGGACTGGAATT
IL-1β	F: AGGATATGGAGCAACAAGT
	R: GCAGGACAGGTACAGATT
IL-6	F: TGGATTCAATGAGGAGACTT
	R: TTCTGGAGGTACTCTAGGTATA
IL8	F: TGCTAAAGAACTTAGATGTCAG
	R: GTCCACTCTCAATCACTCT
IL-17	F: GAAGGCAGGAATCACAATC
	R: GGTTATGGATGTTCAGGTTG
IL-22	F: CCTCAATCTGATAGGTTCCA
	R: CATGTGCTTAGCCTGTTG
TGF-β1	F: GACTACTACGCCAAGGAG
	R: TGTGTACTCTGCTTGAACT
TNF-α	F: AGGGACCTCTCTCTAATCAG
	R: TTTGCTACAACATGGGCTAC

Table 2. IC₅₀, RI, and relative delay rate of drug resistance induced resistant cells.

Stage	Subgroup	IC50 (nmol/L)	Resistance index	Relative delay rate (%)
Parental PC9		12.75 ± 2.14
Stage 1	GEF(10 nmol/L)	20.59 ± 0.25	1.63 ± 0.01
	GEF(10 nmol/L)+ SFI(1 mg/mL)	13.15 ± 0.21	1.04 ± 0.01	36.13
	GEF(10 nmol/L)+ SFI(4 mg/mL)	9.06 ± 0.23	0.72 ± 0.01	56.0
	GEF(10 nmol/L)+ SFI(8 mg/mL)	7.43 ± 0.06	0.58 ± 0.00	63.91
Stage 2	GEF(10 nmol/L)	100.08 ± 1.9	7.8 ± 0.14
	GEF(10 nmol/L)+ SFI(1 mg/mL)	36.54 ± 0.48	2.82 ± 0.03	63.49
	GEF(10 nmol/L)+ SFI(4 mg/mL)	23.41	1.81 ± 0.02	76.61
	GEF(10 nmol/L)+ SFI(8 mg/mL)	18.31 ± 0.05	1.44 ± 0.00	81.70
Stage 3	GEF(10 nmol/L)	383.53 ± 1.8	30.01 ± 0.14
	GEF(10 nmol/L)+ SFI(1 mg/mL)	232.86 ± 10.31	17.33 ± 0.8	39.29
	GEF(10 nmol/L)+ SFI(4 mg/mL)	195.46 ± 1.77	15.2 ± 0.13	49.04
	GEF(10 nmol/L)+ SFI(8 mg/mL)	138.03 ± 0.85	10.82 ± 0.06	64.01
Stage 4	GEF(10 nmol/L)	3988.33 ± 67.5	312.78 ± 5.29
	GEF(10 nmol/L)+ SFI(1 mg/mL)	3766.33 ± 30.23	296.31 ± 2.37	5.57
	GEF(10 nmol/L)+ SFI(4 mg/mL)	3357.33 ± 163.83	248.54 ± 12.84	15.82
	GEF(10 nmol/L)+ SFI(8 mg/mL)	2903 ± 17.57	226.11 ± 1.37	27.21

Data are expressed as mean ± SEM (n = 3).

Figure 1. The delayed drug resistance effect of SFI in NSCLC cells. (A, B, C, D) According to the MTT method, the cell proliferation inhibition rate and IC₅₀ of gefitinib (1, 3, 9, 27, 81, 243, 729, 2187 nmol/L) and RI of each induced drug-resistant cells were measured after 48 h of treatment in four stages. Data are expressed as mean ± SEM (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared to gefitinib alone inducing gefitinib-resistant PC9-GR cells.

Figure 2. SFI relieves gefitinib resistance by reducing IL-22 levels. (A, B) After treating PC9/GR and H1975 cells with SFI (10 mg/mL) for 6 h, the mRNA levels of various cytokines were evaluated. Compared to the control group, * p < 0.05, ** p < 0.01. (C) IL-22 protein expression of cellular protein extracted from indicated cells was assessed by Western blot. *p < 0.05 or **p < 0.01 compared to PC9 cells. (D) Results of qRT-PCR presented IL-22 mRNA levels in PC9/GR and H1975 cells. *p < 0.05 or **p < 0.01 compared to PC9 cells. (E) IL-22 protein expression was verified by Western blot in SFI (10 mg/mL) treated or untreated indicated cells. (F) Cells were treated or untreated with 2.5, 10, or 40 mg/mL SFI for 6 h, then relative IL-22 mRNA level in indicated cells was measured by qRT-PCR. (G) The mRNA expression levels of IL-22RA1 in PC9/GR and H1975 cells after a 6 h treatment of 10 mg/mL SFI were determined using qRT-PCR. (H) The data on the relationship between IL-22 (221165_s_at) expression and overall survival for lung cancer patients and p-values were obtained from the Kaplan-Meier plotter database (http://kmplot.com/analysis/index.php?p=background). (I) After treating PC9/GR and H1975 cells with or without recombinant IL-22 (50 ng/mL) for 48 h, cell proliferation was assessed by MTT assay. **p < 0.01 compared to the control without exogenous addition of IL-22. (J, K) PC9/GR and H1975 cells were incubated with IL-22 (50 ng/mL) at the indicated time points. The relative migration rate was measured. **p < 0.01, ***p < 0.001 compared to the control without exogenous addition of IL-22 at a 48 h time point.

Figure 3. SFI inhibits the STAT3/AKT branch activated by IL-22 treatment in PC9/GR and H1975 cells. (A, B) Western blot of PC9/GR and H1975 cells determining p-STAT3 and p-AKT following stimulation with IL-22 (50 ng/mL) for 10 or 30 min. (C, D) Western blot analysis of PC9/GR and H1975 cells was stimulated with IL-22 (50 ng/mL) for 20 min after pre-treated or not with 10 mg/mL SFI for 24 h or 48 h. Results are expressed as mean ± SEM (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001 when compared with control group; #p < 0.05, ##p < 0.01, ###p < 0.001 when compared with IL-22 group in A, B, C, and D.

Figure 4. SFI facilitates the gefitinib cytotoxicity of PC9/GR and H1975 cells. (A, B) Cell proliferation inhibition profiles were measured after 48 h of different concentrations of gefitinib (30, 15, 7.5, 3.25 μmol/L), respectively co-administered IL-22 (50 ng/mL) alone or in combination with SFI (10 mg/mL) exposure to PC9/GR and H1975 cells. (C) Clonogenic assays of PC9/GR and H1975 cells after gefitinib alone, gefitinib + IL-22, gefitinib + SFI, and gefitinib + IL-22 + SFI treatment for 13 days. (D) After 48 h incubation with different schemes, including gefitinib alone, gefitinib + IL-22, gefitinib + SFI, and gefitinib + IL-22 + SFI, flow cytometric analysis of apoptosis was detected, and the data were processed by FlowJo VX software. Results are expressed as mean ± SEM (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, when compared with gefitinib; ^##p < 0.01, ^###p < 0.001, ^####p < 0.001 when compared with gefitinib + IL-22.

Data availability statement

The data sets used in the current study are available from the corresponding author upon reasonable request.