PRMT5 Selective Inhibitor Enhances Therapeutic Efficacy of Cisplatin in Lung Cancer Cells

Abstract

As a therapeutic approach, epigenetic modifiers have the potential to enhance the efficacy of chemotherapeutic agents. Protein arginine methyltransferase 5 (PRMT5), highly expressed in lung adenocarcinoma, was identified to be involved in tumorigenesis. In the current study, we examined the potential antineoplastic activity of PRMT5 inhibitor, arginine methyltransferase inhibitor 1 (AMI-1), and cisplatin on lung adenocarcinoma. Bioinformatic analyses identified apoptosis, DNA damage, and cell cycle progression as the main PRMT5-associated functional pathways, and survival analysis linked the increased PRMT5 gene expression to worse overall survival in lung adenocarcinoma. Combined AMI-1 and cisplatin treatment significantly reduced cell viability and induced apoptosis. Cell cycle arrest in A549 and DMS 53 cells was evident after AMI-1, and was reinforced after combination treatment. Western blot analysis showed a reduction in demethylation histone 4, a PRMT5- downstream target, after treatment with AMI-1 alone or in combination with cisplatin. While the combination approach tackled lung cancer cell survival, it exhibited cytoprotective abilities on HBEpC (normal epithelial cells). The survival of normal bronchial epithelial cells was not affected by using AMI-1. This study highlights evidence of novel selective antitumor activity of AMI-1 in combination with cisplatin in lung adenocarcinoma cells.

Keywords: A549 and DMS 53; HBEpC; PRMT5; cisplatin; epigenetics; histone; lung cancer.

Figure 1

Bioinformatics analyses to determine the prognostic value of PRMT5 and its major functional pathways in lung cancer (A) Kaplan–Meier survival analysis of overall survival revealed that high PRMT5 expression was significantly associated with worse overall survival (OS) in both lung cancer subtypes of adenocarcinoma and squamous cell carcinoma. (B) Using a publicly available dataset (GSE56757), the differentially expressed genes between PRMT5-silenced and control A549 lung cancer cells were identified and entered into WebGestalt tool to determine the top upregulated and downregulated functional pathways.

Figure 2

AMI-1 alone and in combination with cisplatin reduces the viability of A549 cells. The cellular viability of A549 cells upon treatment with AMI-1.; cisplatin or both was measured using MTT colorimetric assay. (A) A549 cells were exposed to several AMI-1 concentrations (1, 5, and 10 µM) and viability was analyzed at 24, 48 and 72 h. AMI-1 inhibited cellular viability at concentrations of 10 µM when treated for 48 h. (B) Cells were treated with cisplatin alone and in combination with 1, 5 and 10 µM AMI-1., and viability was assessed at 24, 48 and 72 h. 10 μM AMI-1 in combination with IC₅₀ of cisplatin (23.4 µM) significantly inhibited cell survival at 72 h. Data are normalized to untreated control (Ctrl) and is representative of two independent experiments. * represents a statistically significant change in viability between the indicated treatment groups at given time points. ** p < 0.01.; *** p < 0.001.

Figure 3

Protein profiling of PRMT5 and its downstream targets; β-catenin and H4R3me2s.; in A549 cells. A549 cells were treated with AMI-1.; cisplatin or both and harvested after 48 h of treatment. (A) Representative Western blot of PRMT5; β-catenin.; and H4R3me2s levels in A549 cells after administration of AMI-1 and/or cisplatin for 48 h. ß-actin was used as a loading control. AMI-1 (10 μM) in combination with cisplatin markedly reduced the methylation of histone H3. (B) Immunofluorescence staining of A549 cells exposed to different treatment conditions left untreated than were stained for DNA (DAPI; blue).; PRMT5 (green). Arrows indicate subcellular localization of PRMT5 in A549 lung cancer cells under 40× objective lens. (C) Bar graph showing the PRMT5 cellular localization in cells treated with AMI-1 and cisplatin alone or in combination in comparison to untreated cells. (*) Represents the statistically significant change in viability between the indicated treatment groups at given time points. ** p < 0.01.

Figure 4

AMI-1 in combination with cisplatin induces G1 cell cycle arrest in lung cancer cells. The cell cycle progression in A549 and DMS 53 cells upon treatment with AMI-1; cisplatin or both for 48 h was measured by propidium iodide (PI) staining and analyzed using flow cytometry. (A,B) Bar graph showing the percentage of cells in sub-G1; G1; S; and G2/M representative of flow cytometric analysis of A549 cells (A) and DMS 53 cells (B) treated with AMI-1 and cisplatin alone or in combination with cisplatin indicating the different phases of the cell cycle. Percentage of cells occupying sub-G1; G1; S; and G2/M phases of the cell cycle were determined using the cell cycle platform of FlowJo software. Data here are representative of two independent experiments. Tables represent values of individual cell populations in each cell cycle phase. (C) Representative Western blot of cyclin D1; cdk4; and cdk6 levels in A549 cells after administration of AMI-1 and/or cisplatin. ß-actin was used as the loading control.

Figure 5

AMI-1 in combination with cisplatin augments apoptosis in lung cancer cells but not in HBEpC cells. A549; DMS 53 and HBEpC cells were cultured in the presence of AMI-1; cisplatin or both for 72 h. (A) A549 cells were stained with annexin V-FITC/PI and apoptosis read using flow cytometry. Flow cytometric acquired data were analyzed using the FlowJo software. (B) DMS 53 cells were stained with annexin V-FITC/PI and apoptosis read using flow cytometry. (C) HBEpC cells were stained with annexin V-FITC/PI and apoptosis read using flow cytometry Flow cytometric plots demonstrate 10 μM AMI-1 in combination with cisplatin selectively induced apoptosis post 72 h of treatment in both lung cancer cells, not HBEpC cells. Data are from one representative experiment out of at least three. (D) Representative Western blot of caspase-3; PARP; and survivin levels in A549 cells after administration of AMI-1 and/or cisplatin for. ß-actin was used as the loading control.