Baicalein inhibits cell growth and increases cisplatin sensitivity of A549 and H460 cells via miR-424-3p and targeting PTEN/PI3K/Akt pathway

Abstract

Lung cancer is the leading cause of death in individuals with malignant disease. Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer, and chemotherapy drugs such as cisplatin are the most widely used treatment for this disease. Baicalein is a purified flavonoid compound that has been reported to inhibit cancer cell growth and metastasis and increase sensitization to chemotherapeutic drugs via different pathways. Therefore, we assessed the effects of baicalein on the proliferation, apoptosis and cisplatin sensitivity in the NSCLC A549 and H460 cell lines and determined the pathways through which baicalein exerts its effects. Baicalein was slightly toxic to normal human bronchial NHBE cells but inhibited growth, induced apoptosis and increased cisplatin sensitivity in A549 and H460 cells. Baicalein down-regulated miR-424-3p, up-regulated PTEN expression and down-regulated expression of PI3K and p-Akt in A549 and H460 cells. Dual-luciferase reporter assay demonstrated that PTEN is a target gene of miR-424-3p, and overexpression of miR-424-3p or silencing of PTEN partially attenuated the effects of baicalein on A549 and H460 cells. Taken together, we concluded that baicalein inhibits cell growth and increases cisplatin sensitivity to A549 and H460 cells via down-regulation of miR-424-3p and targeting the PTEN/PI3K/Akt pathway.

Keywords: PTEN; baicalein; miR-424-3p; non-small-cell lung cancer.

Figure 1

Cytotoxic effects of baicalein in A549, H460 cells and NHBE cells. (A) Chemical structure of baicalein. (B) NHBE, A549 and H460 cells were treated with different concentrations of baicalein for 24 h, and CCK‐8 was used to detect cell viability of three cell lines. *P < .05

Figure 2

Baicalein inhibits cell proliferation, promotes apoptosis and increases cisplatin sensitivity in A549 and H460 cells via up‐regulation of PTEN and suppression of the PI3K/Akt pathway. (A) A549 and H460 cells were treated with 0 or 40 μmol/L baicalein for 0‐72 h, and CCK‐8 was performed to measure cell proliferation. (B) Clone formation assay was used to detect number of colonies 24 h after baicalein treatment. (C) Annexin V‐FITC/PI double staining and flow cytometry were used to detect apoptosis in A549 and H460 cells treated with 0, 40, 80 μmol/L baicalein for 24 h. (D) Caspase‐3/7 activity assay kit was used to detect caspase‐3/7 activity in A549 and H460 cells. (E) Western blotting was performed to detect expression levels of survivin, Bcl‐xL and proteins involved in the PTEN/PI3K/Akt pathway 48 h after baicalein treatment. (F) Cells treated with 0 or 40 μmol/L baicalein were cotreated with different concentrations of cisplatin for 24 h, CCK‐8 was used to detect cell viability, and the IC50 was calculated. IC50 indicates the concentration of cisplatin at which cell viability is inhibited by 50%. (G,H) Xenograft mice were divided into four groups: vehicle control, baicalein, cisplatin and baicalein combined with cisplatin. Average radiance of each mouse was observed weekly, and tumour weights were recorded at week 4. *P < .05

Figure 3

Baicalein down‐regulates miR‐424‐3p expression in A549 and H460 cells. (A) A549 cells treated with DMSO or 40 μmol/L baicalein for 24 h were collected for RNA extraction and miRNAs microarray analysis. Hierarchical clustering was performed to display the differentially expressed miRNAs with a fold change of ≥2.0 and a P‐value of ≤.05. (B) qRT‐PCR was performed to validate the differential expression of miR‐424‐3p in NSCLC A549 and H460 cells. (C) qRT‐PCR was performed to validate the differential expression of miR‐377‐3p in NSCLC A549 and H460 cells. (D) qRT‐PCR was performed to validate the differential expression of miR‐1224‐5p in NSCLC A549 and H460 cells. *P < .05

Figure 4

PTEN was identified as a target gene of miR‐424‐3p. (A) PTEN mRNA 3′UTR was shown to have a complementary pairing sequence with miR‐424‐3p. (B) miR‐424‐3p mimics, inhibitor and scramble were transfected into A549 and H460 cells for 48 h, and Western blotting was performed to detect the effects of miR‐424‐3p on PTEN protein expression. (C) Dual‐luciferase reporter assay was used to determine the relative luciferase activity of A549 and H460 cells cotransfected with miR‐424‐3p mimics and pmirGLO‐Wt‐PTEN 3′UTR. *P < .05

Figure 5

Effects of miR‐424‐3p down‐regulation or baicalein treatment on cell proliferation, apoptosis and cisplatin sensitivity in A549 and H460 cells. (A) A549 and H460 cells were treated with 40 μmol/L baicalein or transfected with miR424‐3p mimics/scramble. Expression levels of miR‐424‐3p were examined via qRT‐PCR. (B) CCK‐8 was performed to examine the effects on cell proliferation in the three groups at 0‐72 h. (C) Clone formation assay was used to detect colony number in the three groups at 24 h. (D) Annexin V‐FITC/PI double staining and flow cytometry were used to detect apoptotic cells in the three groups at 24 h. E, IC50 of cisplatin was calculated according to the OD values of cells in the three groups at 24 h when cotreated with different concentrations (0, 2, 4, 8, 16, 32 μmol/L) of cisplatin, using the CCK‐8 assay. *P < .05

Figure 6

Overexpression of miR‐424‐3p or silencing of PTEN attenuates the effects of baicalein on A549 and H460 cells. Six groups were designed to determine the pathway of baicalein activity in A549 and H460 cells: Blank, baicalein treatment alone, miR‐424‐3p transfection alone, si‐PTEN transfection alone, baicalein with miR‐424‐3p transfection and baicalein with si‐PTEN transfection. (A) Expression levels of PTEN protein were detected in the six groups via Western blot. (B) Colony number was determined in the six groups via clone formation assay. (C) Apoptosis in cells was detected via Annexin V‐FITC/PI double staining and flow cytometry. (D) IC50 of cisplatin was calculated according to the OD value of cells when cotreated with different concentrations of cisplatin via CCK‐8 assay. *P < .05