Norcantharidin reverses cisplatin resistance and inhibits the epithelial mesenchymal transition of human non‑small lung cancer cells by regulating the YAP pathway

Abstract

Non‑small cell lung cancer (NSCLC) accounts for >80% of all lung cancer cases, which are the leading cause of cancer‑related mortality worldwide. The clinical efficacy of available therapies for NSCLC is often limited due to the development of resistance to anticancer drugs, particularly to cisplatin (DDP). Norcantharidin (NCTD) is a traditional Chinese medicine used in the treatment of many types of cancer, to which patients do not develop resistance. The aim of the present study was to examine the potential synergistic effects of NCTD and DPP on the viability of the the DDP‑resistant NSCLC cell line, A549/DDP. We further explored the potential underlying mechanisms by examining the expression of the oncogene, Yes-associated protein 1 (YAP), whose activation was recently found to be associated with drug resistance. We further examined a series of human lung cancer cell lines and tissues from patients with lung cancer, which revealed that YAP activation contributed to lung cancer initiation, progression and metastasis, and was associated with a poor prognosis, and confering resistance against targeted therapies. Moreover, YAP expression was evaluated in the A549/DDP cells treated with NCTD, DDP, or both drugs. The combined treatment significantly sensitized the A549/DDP cells to DDP‑induced growth inhibition by reducing YAP promoter activity (based on transcriptional expression) and the expression of its target genes, connective tissue growth factor (CTGF) and cysteine rich angiogenic inducer 61 (CYR61). Furthermore, compared to the individual treatments, combined treatment increased cell apoptosis and senescence, and decreased epithelial‑to‑mesenchymal transition and the cell migratory and invasive ability. On the whole, our data indicate that the application of NCTD with reverses DDP resistance and thus, this combined treatment may have promising prospects for use in improving the outcome of patients with NSCLC.

Figure 1.

Aberrant activation of YAP in lung tumors from patients. (A and B) Gel-based qRT-PCR and western blot analysis densitometric quantification demonstrating elevated mRNA and protein expression levels of YAP in human non-small cell lung cancer (NSCLC) cells compared with the normal HBEC cells. (C) CCK-8 assay demonstrated that the NSCLC cells had a greater proliferative capability than the HBEC cells. (D) Immunohistochemical staining of YAP proteins showing increased total levels of YAP and that more YAP was accumulated in the nucleus in NSCLC samples compared with their normal adjacent lung tissues, while more YAP was localized in the cytoplasm of the normal adjacent lung tissues. (E) Relative protein and mRNA levels of YAP were assessed by western blot analysis and RT-qPCR in 20 pairs of NSCLC tissues and corresponding non-tumor tissues. (F) Kaplan-Meier overall survival (OS) curves of YAP (n=1926, p=1E-09 by log-rank test for significance). Results are presented as the means ± SD, and the error bars represent the SD of 3 independent experiments. ***P<0.001 vs. normal cells or tissues.

Figure 2.

YAP regulates non-small cell lung cancer (NSCLC) cell growth and invasion. (A) RT-PCR and western blot analysis showing the mRNA and protein expression of YAP following transfection of A549 cells with siRNA against YAP or YAP overexpression vector. (B) CCK-8 assay demonstrating that the knockdown of YAP using siRNA or the stable overexpression of YAP significantly suppressed or promoted the proliferation of A549 cells, respectively. (C) Colony formation assay demonstrated that the colony formation density was significantly increased in cells stably expressing YAP and decreased in cells in which YAP was knocked down using the siRNA. (D) SA-β-Gal assay showing that the stable overexpression or knockdown of YAP using siRNA significantly blocked and promoted the cell senescence phenotype, respectively. (E) Scratch assay indicated that the stable overexpression of YAP or the knockdown of YAP using siRNA markedly increased or decreased the migration of A549 cells for 36 h, respectively. (F) Transwell assay identified that the stable overexpression of YAP or the knockdown of YAP using siRNA significantly increased or decreased cell invasive growth compared with the A549 control cells. Results are presented as the means ± SD, and the error bars represent the SD of 3 independent experiments. *P<0.05; **P<0.01; ***P<0.001 vs. the control group.

Figure 3.

Norcantharidin (NCTD) sensitizes cells to cisplatin (DDP)-induced tumor growth inhibition. (A) RT-PCR and western blot analysis revealed that the mRNA and protein expression levels of YAP and its targets genes, including CYR61 and CTGF were much higher in the A549/DDP and H1299/DDP cells than in the parent A549 and H1299 cells. (B and C) CCK-8 and MTT assay indicated that a higher, but not lower concentration of NCTD suppressed A549/DDP and H1299/DDP cell proliferation. However, the higher concentration of DDP still did not suppress A549/DDP cell proliferation. (D) Cellular growth and proliferation assay showing that a low dose of NCTD sensitized the A549/DDP and H1299/DDP cells to the anti-proliferative effects of low doses of DDP. Results are presented as the means ± SD, and the error bars represent the SD of 3 independent experiments. *P<0.05; **P<0.01; ***P<0.001 vs. control group. NS, not significant.

Figure 4.

Norcantharidin (NCTD) sensitizes non-small cell lung cancer (NSCLC) cells to cisplatin (DDP) and suppresses the activity of YAP. (A) The effect of NCTD, DDP or co-treatment with both agents on the activities of YAP promoter was examined by luciferase reporter gene assays in A549/DDP and H1299/DDP cells. (B and C) RT-qPCR and western blot analysis demonstrating that NCTD enhanced the DDP-induced suppressive effects on the YAP, CTGF and CYR61 on mRNA and protein levels in the A549/DDP cells. (D) Immunofluorescence staining of YAP protein demonstrating that NCTD enhanced the DDP-induced suppressive effects on YAP expression in the A549/DDP cells. Results are presented as the means ± SD, and the error bars represent the SD of 3 independent experiments. *P<0.05; ***P<0.001 vs. the control group. NS, not significant.

Figure 5.

Norcantharidin (NCTD) sensitizes lung cancer cells to cisplatin (DDP)-induced senescence and apoptosis. (A) Phase contrast microscopy revealed that cellular morphology was significantly altered, from a flat to round shape, by co-treatment with NCTD and DDP in A549/DDP cells, which indicated that cells were in a state of poor survival. (B) Colony formation assay demonstrated that the colony formation density was significantly decreased following co-treatment with NCTD and DDP in A549/DDP cells. (C) SA-β-Gal assay showing that NCTD enhanced the DDP-induced cell senescence phenotype in A549/DDP cells. (D) Western blot analysis and densitometric quantification demonstrating that NCTD enhanced DDP-induced the expression of active caspase-3 on protein level in A549/DDP cells. (E and F) Flow cytometric and immunofluorescent staining assay demonstrating that NCTD enhanced the DDP-induced apoptosis of A549/DDP cells. Results are presented as the means ± SD, and the error bars represent the SD of 3 independent experiments. ***P<0.001 vs. control group. NS, not significant.

Figure 6.

Norcantharidin (NCTD) sensitizes non-small cell lung cancer (NSCLC) cells to cisplatin (DDP) and suppresses YAP-mediated invasiveness and epithelial-mesenchymal transition (EMT). (A) RT-PCR and western blot analysis showing the mRNA and protein expression of E-cadherin and vimentin in A549/DDP cells transfected with siRNA or YAP overexpression vector. (B and C) RT-qPCR and western blot analysis and densitometric quantification demonstrating that NCTD enhanced the DDP-induced increase or decrease in the (B) mRNA and (C) protein level of E-cadherin or vimentin in A549/DDP cells. (D) Scratch assay showing that NCTD enhanced the DDP-induced decrease in the migration of A549/DDP cells at 36 h. (E) Transwell assay identified that NCTD enhanced the DDP-induced decrease in cell invasive growth and migration of the A549/DDP cells. (F) The correlation between the co-treatment with NCTD and DDP and EMT marker protein levels, E-cadherin and vimentin, was analyzed based on the results of western blot analysis, respectively. Results are presented as the means ± SD, and the error bars represent the SD of 3 independent experiments. ***P<0.001 vs. the control group. NS, not significant.

Figure 7.

Schematic diagram of the mechanisms through which norcantharidin (NCTD) reverses cisplatin resistance and inhibits the epithelial mesenchymal transition of human cisplatin-resistant lung cancer cells via regulating the YAP pathway. NCTD may thus be an effective compound which may be used to reverse the resistance of human lung cancers to DDP by inhibiting YAP-induced anti-apoptotic effects, epithelial-mesenchymal transition (EMT), and proliferation and invasiveness.