Luteolin enhances drug chemosensitivity by downregulating the FAK/PI3K/AKT pathway in paclitaxel‑resistant esophageal squamous cell carcinoma

Abstract

Drug resistance is a key factor underlying the failure of tumor chemotherapy. It enhances the stem‑like cell properties of cancer cells, tumor metastasis and relapse. Luteolin is a natural flavonoid with strong anti‑tumor effects. However, the mechanism(s) by which luteolin protects against paclitaxel (PTX)‑resistant cancer cell remains to be elucidated. The inhibitory effect of luteolin on the proliferation of EC1/PTX and EC1 cells was detected by cell counting kit‑8 assay. Colony formation and flow cytometry assays were used to assess clonogenic capacity, cell cycle and apoptosis. Wound healing and Transwell invasion tests were used to investigate the effects of luteolin on the migration and invasion of EC1/PTX cells. Western blotting was used to detect the protein levels of EMT‑related proteins and stem cell markers after sphere formation. Parental cells and drug‑resistant cells were screened by high‑throughput sequencing to detect the differential expression of RNA and differential genes. ELISA and western blotting were used to verify the screened PI3K/Akt signaling pathway, key proteins of which were explored by molecular docking. Hematoxylin and eosin staining and TUNEL staining were used to observe tumor xenografts on morphology and apoptosis in nude mice. The present study found that luteolin inhibited tumor resistance (inhibited proliferation, induced cell cycle arrest and apoptosis and hindered migration invasion, EMT and stem cell spherification) in vitro in PTX‑resistant esophageal squamous cell carcinoma (ESCC) cells. In addition, luteolin enhanced drug sensitivity and promoted the apoptosis of drug‑resistant ESCC cells in combination with PTX. Mechanistically, luteolin may inhibit the PI3K/AKT signaling pathway by binding to the active sites of focal adhesion kinase (FAK), Src and AKT. Notably, luteolin lowered the tumorigenic potential of PTX‑resistant ESCC cells but did not show significant toxicity in vivo. Luteolin enhanced drug chemosensitivity by downregulating the FAK/PI3K/AKT pathway in PTX‑resistant ESCC and could be a promising agent for the treatment of PTX‑resistant ESCC cancers.

Keywords: drug chemosensitivity; esophageal squamous cell carcinoma; focal adhesion kinase/PI3K/AKT pathway; luteolin; molecular docking.

Figure 1

The effects of PTX on the proliferation of EC1 and EC1/PTX cells. (A) The inhibitory effects of PTX at different concentrations (0.005, 0.01, 0.02, 0.04, 0.08, 0.16 μg/ml) on the proliferation of EC1 cells. (B) The inhibitory effects of PTX at different concentrations (0.05, 0.1, 0.2, 0.4, 0.8, 0.16 μg/ml) on the proliferation of EC1/PTX cells. ^*P<0.05, ^**P<0.01, ***P<0.001 vs. DMSO. PTX, paclitaxel.

Figure 2

Effects of luteolin on cell proliferation, cell cycle and apoptosis in PTX-resistant ESCC cells. (A) The inhibitory effects of luteolin at different concentrations (10, 20, 40, 80 and 120 μM) on the proliferation of EC1 cells and EC1/PTX drug-resistant cells. (B) Luteolin (10, 20 and 40 μM) exerted its effects on EC1/PTX cells. After 7 days, the cells were stained with 0.1% crystal violet solution and clone proliferation was analyzed. (C) Luteolin (10, 20 and 40 μM) acted on EC1/PTX cells for 48 h. The apoptosis rate of cells in different treatment groups was determined by flow cytometry after Annexin-FITC/PI staining. (D) Luteolin (10, 20 and 40 μM) was used to treat EC1/PTX cells for 24 h and cell cycle distribution was determined by flow cytometry after PI staining. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. DMSO n=3. PTX, paclitaxel; ESCC, esophageal squamous cell carcinoma.

Figure 3

Effects of luteolin on the migration, invasion, EMT and dry spheroidization of PTX-resistant ESCC cells. (A) Wound healing assay was used to evaluate the inhibitory effect of luteolin on the migration potential of EC1/PTX cells (magnification, ×200). (B) A Transwell chamber was used to determine the inhibitory effect of luteolin on the invasion potential of EC1/PTX cells (magnification, ×100). (C) TGF-β1 was used to induce EMT in EC1/PTX cells and the cells were treated with 10 or 20 μM luteolin. The expression levels of EMT-related proteins were determined by western blotting. (D) A cell microsphere formation test was performed to determine the effect of luteolin on the spheroidizing potential of EC1/PTX cells (magnification, ×50). (E) After EC1/PTX cells were treated with luteolin at different concentrations (10, 20 and 40 μM) for 24 h, western blotting was performed to determine the expression levels of stem cell markers in the cells. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. DMSO n=3. EMT, epithelial-mesenchymal transition; PTX, paclitaxel; ESCC, esophageal squamous cell carcinoma.

Figure 4

A combination of luteolin and PTX increased drug sensitivity and apoptosis in PTX-resistant ESCC. (A) The survival rate of EC1/PTX cells after 24 h of treatment with luteolin (10, 20 and 40 μM). (B) The effects of luteolin on the PTX sensitivity of EC1/PTX cells. ^***P<0.001 vs. DMSO. (C) Luteolin, PTX and luteolin plus PTX were used to treat EC1/PTX cells for 48 h. After Annexin-FITC/PI staining, the apoptosis rate of cells in the different treatment groups was determined by flow cytometry. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. Control, ^##P<0.01, ^###P<0.001 vs. PTX, n=3. PTX, paclitaxel; ESCC, esophageal squamous cell carcinoma.

Figure 5

KEGG and GO analysis of the aberrant expression of genes. (A) A heatmap and volcano map were prepared by next-generation sequencing. (B) KEGG biological pathway enrichment analysis. (C) GO gene function enrichment analysis. KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology.

Figure 6

The expression patterns of proteins associated with the FAK/Src/PI3K/Akt pathway and PTX resistance in ESCC cells. Luteolin (10, 20 and 40 μM) was used to treat EC1/PTX cells for 24 h. (A) Proteins associated with the FAK/Src/PI3K/Akt pathway were detected by western blotting. (B) Drug resistance-associated proteins were detected by western blotting. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. DMSO n=3. FAK, focal adhesion kinase; PTX, paclitaxel; ESCC, esophageal squamous cell carcinoma; p-, phosphorylated; MRP1, multidrug resistance protein 1; BCRP, breast cancer resistance protein.

Figure 7

Visualization of the molecular docking of luteolin with target proteins. Molecular modeling was used to dock luteolin into the binding sites of (A) FAK, (B) Src and (C) AKT. FAK, focal adhesion kinase.

Figure 8

Effect of treatment with luteolin combined with PTX on the xenografts in nude mice and apoptosis in vivo. Luteolin combined with PTX was used to treat xenografts in nude mice. (A) The body weights of nude mice were measured. (B) The tumor volumes were measured. (C) The images of tumors were acquired. (D) The average tumor mass was measured. ^**P<0.01 vs. NC, ^#P<0.05 vs. 20 mg/kg, n=5. (E) Hematoxylin and eosin staining assay for cell morphology in different groups (magnification, ×100). (F) TUNEL assay for apoptosis in different groups (magnification, ×50). ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. NC, ^#P<0.05, ^##P<0.01 vs. PTX + LUT, n=5. PTX, paclitaxel; LUT, luteolin; NC, normal control.

Figure 9

Effect of luteolin on the expression patterns of proteins associated with the FAK/Src/PI3K/Akt pathway and drug resistance in vivo. Luteolin (20 or 40 mg/kg) was used to treat xenografts in nude mice. (A) Proteins associated with the FAK/Src/PI3K/Akt pathway were detected by western blotting. (B) The resistance-associated proteins were detected by western blotting. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. NC, n=3. FAK, focal adhesion kinase; PTX, paclitaxel; ESCC, esophageal squamous cell carcinoma; p-, phosphorylated; MRP1, multidrug resistance protein 1; BCRP, breast cancer resistance protein; NC, normal control.