Matrine increases the inhibitory effects of afatinib on H1975 cells via the IL‑6/JAK1/STAT3 signaling pathway

Abstract

Resistance to epidermal growth factor receptor (EGFR) inhibitors is of primary concern in the treatment of non‑small‑cell lung cancer (NSCLC) with EGFR mutations. To investigate the effects of matrine on H1975 cells and to examine a novel, potential treatment option for NSCLC, the present study measured cell viability, apoptotic rate, interleukin 6 (IL‑6) expression and activation of the janus kinase (JAK) 1/signal transducer and activator of transcription (STAT)3 signaling pathway in cells treated with or without matrine, in the presence or absence of afatinib. The results demonstrated that matrine treatment inhibited cell growth, decreased B‑cell lymphoma 2 (Bcl‑2) expression and induced apoptosis. Matrine treatment additionally decreased the mRNA and protein levels of IL‑6 and inhibited activation of the JAK1/STAT3 signaling pathway in H1975 cells in a dose‑dependent manner. H1975 cells treated with IL‑6 small interfering RNA exhibited a decrease in Bcl‑2 expression levels and cell viability. Treatment with a combination of matrine and afatinib demonstrated increased inhibitory effects on the growth rate of H1975 cells. The findings of the present study suggested that matrine treatment decreases IL‑6 expression, inhibits activation of the JAK1/STAT3 signaling pathway, reduces the expression levels of Bcl‑2 and inhibits cell growth. Furthermore, matrine treatment was demonstrated to increase the inhibitory effects of afatinib on H1975 cells with the T790M EGFR mutation.

Figure 1.

Effects of matrine treatment on viability of H1975 cells. (A) Structure of matrine. (B) Cell viability was measured using a Cell Counting Kit-8 (CCK-8) assay and shown by the absorbance at 450 nm after treatment with various concentrations of matrine for 24 h. Data are representative of three independent experiments and expressed as means ± standard error of the mean (SEM). ***P<0.001 compared with control.

Figure 2.

Effects of matrine treatment on apoptosis in H1975 cells. (A) Apoptosis was detected by Annexin V-FITC and propidium iodide (PI) staining in H1975 cells treated with matrine. (B) Graph of (A). (C) Protein levels of Bcl-2, cleaved caspase-3, and total caspase-3 were detected by western blot analysis in H1975 cells treated with various concentrations of matrine for 24 h. β-actin is included as a loading control. Data are representative of three independent experiments and expressed as means ± SEM. ***P<0.001 compared with control.

Figure 3.

Effects of matrine treatment on the JAK1/STAT3 signaling pathway and IL-6 levels in H1975 cells. (A) Protein levels of p-JAK1 and p-STAT3 in H1975 cells treated with various concentrations of matrine for 24 h were detected by western blot analysis. (B) mRNA expression of IL-6 in H1975 cells treated with matrine for 24 h was detected using real-time RT-PCR. (C) Protein levels of IL-6 from the supernatant of H1975 cells treated with different concentrations of matrine were detected by enzyme-linked immunosorbent assay (ELISA). β-actin was included as an mRNA and protein loading control. Data are representative of three independent experiments and expressed as means ± SEM. **P<0.01 and ***P<0.001 compared with control.

Figure 4.

Effects of matrine treatment on the IL-6/JAK1/STAT3 signalign pathway in H1975 cells treated with IL-6 siRNA. (A) Transcriptional expression of IL-6 in H1975 cells treated with or without IL-6 siRNA was detected by real-time RT-PCR. (B) Protein levels of IL-6 from the supernatant of H1975 cells treated with or without IL-6 siRNA were detected by ELISA. (C) Protein levels of p-STAT3 and Bcl-2 in H1975 cells treated with or without IL-6 siRNA in the presence or absence of 2 mM matrine were detected by western blot. β-actin was included as an mRNA and protein loading control. Data are representative of three independent experiments and expressed as means ± SEM.

Figure 5.

Effects of matrine treatment on apoptosis and viability of H1975 cells treated with IL-6 siRNA. (A) Apoptosis was determined by Annexin V-FITC and PI staining in H1975 cells treated with 2 mM matrine or IL-6 siRNA. (B) Graph of (A). (C) The inhibitory effects of IL-6 siRNA on the growth of H1975 cells treated with or without 2 mM matrine were measured with the CCK-8 assay and shown by the absorbance at 450 nm. Data are representative of three independent experiments and expressed as means ± SEM. **P<0.01 and ***P<0.001 compared with control. ^#P<0.05 compared with the group treated with matrine in the absence of IL-6 siRNA.

Figure 6.

Effects of afatinib treatment on apoptosis and the IL-6/JAK1/STAT3 signaling pathway in H1975 cells. (A) Cell viability was measured with the CCK-8 assay and shown by the absorbance at 450 nm following treatment with various concentrations of afatinib for 24 h. (B) Protein levels of IL-6 from the supernatant of H1975 cells treated with or without 1 µM afatinib were detected by ELISA. (C) Activation of the JAK1/STAT3 signaling pathway was detected in H1975 cells in the presence or absence of 1 µM afatinib by western blot. β-actin was included as an mRNA and protein loading control. Data are representative of three independent experiments and expressed as means ± SEM. **P<0.01, and ***P<0.001 compared with control.

Figure 7.

Effects of matrine and afatinib treatment on the growth of H1975 cells in vitro. (A) Protein levels of IL-6 from the supernatant of H1975 cells treated with or without 1 µM afatinib in the presence or absence of 2 mM matrine were detected by ELISA. (B) Protein levels of p-JAK1 and p-STAT3 were detected by western blot prepared from H1975 cells incubated with 1 µM afatinib combined with 2 mM matrine. (C) Effects of of afatinib and matrine treatment on the growth of H1975 cells were measured with the CCK-8 assay and shown by the absorbance at 450 nm. β-actin was included as an mRNA and protein loading control. Data are representative of three independent experiments and expressed as means ± SEM. *P<0.05, **P<0.01, and ***P<0.001 compared with control. ^##P<0.01 compared with the group treated with afatinib in the absence of matrine.

Figure 8.

Effects of matrine and afatinib treatment on the growth of H1975 cells in vivo. After 5×10⁶ cells were injected subcutaneously under the back skin of 5-to-6-week-old male BALB/c nude mice, mice were treated with or without matrine (5 g/kg) in the presence or absence of afatinib (5 mg/kg) formulated in saline by intraperitoneal injection once daily for 4 weeks and tumor volume was estimated once every three days with the formula: volume = l × w² × 0.536, where l and w are perpendicular measured diameters. Data are expressed as means ± SEM. *P<0.05, **P<0.01.