Activation of AIFM2 enhances apoptosis of human lung cancer cells undergoing toxicological stress
Abstract
Application of cisplatin (DDP) for treating lung cancer is restricted due to its toxicity and lung cancer’s drug resistance. In this study, we examined the effect of Jinfukang (JFK), an effective herbal medicine against lung cancer, on DDP-induced cytotoxicity in lung cancer cells. Morphologically, we observed that JFK increases DDP- induced pro-apoptosis in A549 cells in a synergistic manner. Transcriptome profiling analysis indicated that the combination of JFK and DDP regulates genes involved in apoptosis-related signaling pathways. Moreover, we found that the combination of JFK and DDP produces synergistic pro-apoptosis effect in other lung cancer cell lines, such as NCI-H1975, NCI-H1650, and NCI-H2228. Particularly, we demonstrated that AIFM2 is activated by the combined treatment of JFK and DDP and partially mediates the synergistic pro-apoptosis effect. Collectively, this study not only offered the first evidence that JFK promotes DDP-induced cytotoxicity, and activation of AIFM2 enhances apoptosis of human lung cancer cells undergoing toxicological stress, but also provided a novel insight for improving cytotoxicity by combining JFK with DDP to treat lung cancer cells.
Introduction
Lung cancer is the leading cause of cancer-related deaths in the world.Histologically, it consists mainly of squamous-cell carcinoma and adenocarcinoma (Li and Hong, 2013; Zakowski, 2015). Systemic chemotherapy of lung cancer includes conventional chemotherapeutic agents, inhibitors of angiogenesis, and inhibitors of metastasis (Siegel et al., 2013). Although chemotherapeutic advances have been made over the past 20 years, the effects of these chemotherapeutic drugs are transient, and theoverall five-year survival rate is less than 15% (Siegel et al., 2013). Currently, the major challenge for lung cancer therapy is to enhance the efficacy of first-line chemotherapy drugs and develop novel therapeutic agents that can complement the present chemotherapeutic limitation.Platinum compounds are the foundation of chemotherapy regimens for human lung cancer (Pfister et al., 2004). Platinum compounds induce DNA inter-strand crosslinking and inhibit transcription and DNA replication in cancer cells (Gately and Howell, 1993; Kosmider et al., 2004; Wozniak et al., 2004). Cisplatin (DDP), a first-line chemotherapy drug for the treatment of human lung cancer, has poor response rates due to the development of drug resistance (Gao et al., 2013). Several studies demonstrated that the poor response rates and limited response duration of DDP result from down-regulation of multiple apoptosis-related signaling pathways (Galluzzi et al., 2012; Siddik, 2003). In addition, dosage of DDP is clinically limited due to its toxicity. Therefore, it is necessary to find an adjuvant agent that could increase the efficacy of DDP against lung cancer.Traditional Chinese medicine (TCM) has been effectively used against diseases for thousands of years in China. Studies have shown that combinations of herbal extracts and DDP produce a synergistic, tumor-targeted cytotoxicity effect (Berkovich et al., 2013; Chen et al., 2015; Li et al., 2009; Zhao et al., 2011). The Jinfukang (JFK) formula in herbal medicine has been used to treat lung cancer for more than ten years (Cassileth et al., 2009; Jiao et al., 2015; Liu et al., 2007; Liu et al., 2001; Lu et al., 2016a; Lu et al., 2016b). However, the combined effect of JFK and DDP on lung cancer cells has not been analyzed. To address this issue, in the present study, we evaluated the combined effect of JFK and DDP on human lung cancer cell lines and investigated the underlying molecular mechanisms involved.
2. Materials and methods
Procedures of JFK extract preparation were performed according to our previous study (Lu et al., 2016a). Briefly, the JFK formula contains 12 herbs in specified proportions. Raw herbs, including Astragalus membranaceus, Glehnia littoralis, Asparagus cochinchinensis et al. (Lu et al., 2016a), were minced and extracted with 200 milliliters (ml) of 70% ethanol at 80 °C for 1 hour (h). The extraction of JFK was filteredthrough a 0.45 micrometer (μm) syringe filter, evaporated to dryness, dissolved in EtOH to a concentration of 30 mg/ml, and stored at –20 °C until it was used. DDP was purchased from Qilu Pharmaceutical Co., Ltd., China. Initially, DDP was dissolved to a concentration of 6 mg/ml and stored at –20 °C until it was used.Procedures for cell culture were performed according to our previous study (Lu et al., 2016b). Briefly, the human lung cancer cell lines A549, NCI-H1975, NCI-H1650, and NCI-H2228 were obtained from the Shanghai Institute of Biochemistry and cell biology. Cells were cultured in RPMI 1640 medium (Gibco, USA) and incubated at 37°C in a humidified atmosphere of 5% CO2. Cultures were supplemented with 10% fetal bovine serum (FBS) (Gibco, USA). Penicillin-streptomycin (Life Technologies, Inc.USA) was added in the culture media in a concentration of 100 units potassium penicillin and 100 microgram (μg) streptomycin sulfate per 1 ml.Cell counting kit 8 (CCK8) (Dojindo, Japan) was used to assess cell viability in human lung cancer cell lines according to the manufacturer’s protocol. Briefly, 1500 A549 cells were cultured in each well of 96-well plates overnight and incubated with JFK (10 μg/ml, 20 μg/ml and 30 μg/ml, respectively) and DDP (6 μg/ml), alone or together, for indicated time set. For NCI-H1975, NCI-H1650, and NCI-H2228 cells, cell viabilities were examined by CCK8 after exposing these cell lines to JFK (30 μg/ml) and DDP (6 μg/ml), alone or together, for 24 h, 48 h and 72h, respectively. Ten microliters (μL)CCK-8 in 100 μL RPMI 1640 medium was added to each well containing different drug- treated human lung cancer cell samples and incubated for 2 h at 37 °C.
Cell culture viabilities were calculated by measuring the optical density at 450 nm, using a spectrophotometric plate reader (Bio-Tek, USA). Absorbance of the control sample was set at 100% viability, and absorbance of cell-free wells containing medium was set at zero.A549 (1 × 105 cell/well) cells were seeded on 6-well plates and incubated with JFK (30 μg/ml) and DDP (6 μg/ml) for 48 h, alone or together. Each sample was stained with Propidium iodine (PI) (Sigma, USA) and observed by fluorescence microscopy (Nikon, Japan). Three types of pictures were obtained through different excitation wavelengths. Cells were designated death if they were highly red, non-death if staining was slight or none.A549 (1 × 105) cells were cultured in each well of 6-well plates and incubated with JFK (30 μg/ml) and DDP (6 μg/ml) for 48 h, alone or together. Cell cultures were fixed in 4% paraformaldehyde, incubated in Apo-Green labeling mix (Vazyme Biotech Co., Ltd,China) and Hoechst33324 solution (Sigma, USA), and then observed by fluorescence microscope (Nikon, Japan). Cell nucleus was designated DNAfragmentation if the staining was highly green, non-DNA fragmentation if the staining was slight or none.
2.6. Apoptosis analysisAnnexin V-FITC/PI Apoptosis kit (Zoman Biotechnology Co., Ltd, China) was used to determine the phosphatidyl serine and membrane integrity. AIFM2 knockdown or non- AIFM2 knockdown A549, NCI-H1975, NCI-H1650, and NCI-H2228 cells, respectively, were cultured in 6-well plates and incubated with JFK (30 μg/ml) and DDP (6 μg/ml) for 48 h, alone or together. Cells were stained with PI and Annexin V FITC simultaneously and then analyzed by flow cytometry (BD LSRFortessa, USA). PI-positive cells were designated end-stage apoptotic cells, and FITC-positive cells were designated early stage- apoptotic cells.2.7.
RNA-seq library constructionRNA-seq–based transcriptome profiling was performed to assess differential genes, using Illumina HiSeqTM 2000 platform (Illumina, USA). Briefly, A549 cells were exposed to JFK (30 μg/ml) and DDP (6 μg/ml) for 48 h, alone or together. Total RNA of each sample was extracted by Trizol (Life Technologies, Inc., USA) according to the manufacturer’s protocol. mRNA was isolated via Oligotex mRNA Mini Kit (Qiagen, Germany) and then treated with DNase for removing residual genomic DNA. One hundred nanograms (ng) mRNA of each sample was fragmented and end-repaired and adapters ligated using NEBNext Ultra Directional RNA Library Prep Kit (NEB, USA).Each sample was amplified 12 cycles in a thermal cycler, using Q5 High-Fidelity DNA Polymerase (NEB, USA) and corresponding PCR Master Mix. Last, the PCR product was quantified using Nanodrop (Thermo, USA), performed by standard single-end sequencing with 50 bp reads. The raw sequencing data of this study are available in the EMBL database under accession number E-MTAB-4671: http://www.ebi.ac.uk/arrayexpress/.
2.8. Analysis of RNA-seq raw dataThe raw sequencing reads were extracted and analyzed by Illumina software.Sequencing quality was detected by FastQC software. All qualified sequence tags were mapped to reference genome (hg19) by TopHat (Trapnell et al., 2012). Cufflinks was used for characterizing the differential transcription pattern (Trapnell et al., 2012). Biases in library preparation have been taken into account. Gene expression level was measured by reads per kilo-base of transcript per million reads mapped (RPKM). For analyzing the gene expression difference between administration samples and the control sample, we picked out the genes whose gene expression level was at least two-fold change.According to the analytical method, the log2Fold Change > 1 represented the gene expression level of at least two-fold upregulation, and the log2Fold Change < –1 represented the gene expression level of at least two-fold downregulation.
Analysis of gene function cluster and pathway cluster were performed by a public bioinformatics resource platform named Database for Annotation, Visualization and Integrated Discovery (DAVID) (Dennis et al., 2003). Briefly, a list of differentially expressed genes was uploaded to DAVID Bioinformatics Resources 6.7 and performed analysis of the gene ontology (GO) cluster and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway cluster. Besides this, network construction was performed by uploading GO terms to Reduce Visualize Gene Ontology (REVIGO, http://revigo.irb.hr/), and then a network graph was modified using Cytoscape software. Total RNA extraction was performed as mentioned. Reverse transcription was performed using random primer and Super Scrip®III Reverse Transcriptase (18080-044,Invitrogen) according to the manufacturer's protocol. The transcription level of the gene of interest was examined by quantitative real-time PCR (RT-qPCR) using ABI step one plus (Applied Biosystems, USA). GAPDH was used as a control gene for normalization. The relative level of mRNA was calculated as 2−ΔΔCt. All primer sequences used for RT- qPCR are shown in Table S1.2.11. RNA interference RNA interference assays were performed according to our previous study (Qi et al., 2015). Cells were transfected with AIFM2 siRNA (AIFM2Ⅰ: 5’- CCAAAUCAGUGGCUUCUAUTT-3’; AIFM2Ⅱ: 5’-GCACCGGCAUCAAGAUCAATT-3’; AIFM2Ⅲ: 5‘-GCUGCCUCUCAAUGAGUAUTT-3’) when they reached 50% confluence, using Lipofectamine 3000 reagent (Invitrogen). An unrelated, scrambled siRNA was used as negative control (5’-UUCUCCGAACGUGUCAGGUTT-3’).
Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.5% v/v Triton x-100, 0.5% w/v Deoxycholate, 0.1% SDS) and subjected to western blot analysis according to standard techniques. Briefly, the protein samples (~60 µg) were fractionated by SDS-PAGE (12% polyacrylamide gels). Proteins were transferred to a nitrocellulose membrane at moist conditions. Primary antibody against AIFM2 (1:1000, Thermo, USA) was used for binding AIFM2 protein specifically, and the primary antibody against GAPDH (1:2000, CST, USA) was used as an internal control. The protein–antibody complexes were detected using goat-rabbit horseradish peroxidase-conjugated secondary antibodies (1:15000, CST, USA). Images were caught and analyzed by use of LI-COR Odyssey Infrared Imaging system (LI-COR, USA).Data are presented as the mean ± SD. The differences between the samples were examined using the standard one-way ANOVA. SPSS for Windows 14.0 software package was used. p-values were calculated using two-tailed Student’s t-test. Differences were considered significant at P < 0.05. All experiments, excluding RNA-seq and western blot, had at least three replicates. 3. Results cancer cell line A549 with JFK and DDP, alone or together, and assessed the cytotoxicity by CCK-8 assay. Based on our previous study (Lu et al., 2016a), A549 cells were treated with JFK (10 μg/ml, 20 μg/ml, and 30 μg/ml, respectively) and DDP (6 μg/ml) for 48 h, alone or together. Cell viability examination suggests that the synergistic manner of proliferation inhibition presented significantly after A549 cells were exposed to JFK (20 μg/ml and 30 μg/ml, respectively) plus DDP (6 μg/ml) for 48 h (Fig. 1A, *P < 0.001).Time-dependent assays suggest that the synergistic manner presented markedly when A549 cells were exposed to JFK (30 μg/ml) plus DDP (6 μg/ml) for more than 48 hours (Fig. 1B, *P < 0.001). As to apoptosis, a large number of A549 cells are remarkably induced to apoptosis in a synergistic manner after being exposed to JFK and DDP simultaneously for 48 h (Fig. 1C, *P < 0.001). The synergistic pro-apoptosis manner induced by combined treatment with JFK and DDP not only presents in early apoptosis, but also presents in total apoptosis (Fig. 1D and E, *P < 0.01). Compared to JFK and DDP–induced A549 cell death, respectively, JFK plus DDP–induced dying cells are largely increased after treatment for 48 h (Fig. 1F, *P < 0.05; Fig. S1). Besides, TUNEL assays also suggest a synergistic pro-apoptosis manner induced by JFK combined with DDP in A549 cell has been expanded to DNA fragmentation (Fig. 1G).To understand further the molecular mechanism of synergistic pro-apoptosis induced by combined treatment with JFK and DDP in an A549 cell, transcriptome profiling of A549 cells treated with Control, JFK, DDP, and JFK plus DDP, respectively, was performed using RNA-seq. Transcriptome analysis indicates that 1,529 genes (log2Fold Change < –1) were downregulated, and 1,897 genes (log2Fold Change > 1) were upregulated after A549 was exposed to JFK and DDP simultaneously for 48 h (Table S2). A heat map of significantly modulated genes (log2Fold Change < –1 and log2Fold Change > 1) indicates a dominant role for JFK and DDP in A549 cells in affecting gene expression levels (Fig. 2A).Gene ontology (GO) analysis of differentially expressed genes (log2Fold Change < –1 and log2Fold Change > 1) indicates that multiple biological processes are involved in cell apoptosis when A549 cells are treated with JFK and DDP for 48 h, alone or together (Table 1; Table S3 and S4). Among all the GO terms that were modulated by the combination of JFK and DDP, 253 downregulated and 68 upregulated GO terms are specifically affected in A549 cells (Fig. 2B). Furthermore, KEGG pathway analysis of the differentially expressed genes indicates that multiple signal pathways are suppressed or activated when A549 cells were treated with JFK and DDP simultaneously (Fig. S2).To determine the molecular mechanisms involved in and/or regulated by the synergistic pro-apoptosis activity of JFK plus DDP treatment, we compared those differential genes (log2Fold Change < –1 and log2Fold Change > 1) to the cancer gene pool (http://www.bushmanlab.org/links/genelists).
In total, 351 cancer genes are modulated by combined treatment of JFK and DDP in A549 cells, including 228 downregulated cancer genes and 123 upregulated cancer genes (Fig. 2C; Table S2). Bioinformatics analysis reveals that these 351 differentially expressed cancer genes are primarily involved in apoptosis-related signaling pathways or biological processes (Table 2; Fig. S3).Next, 126 synergistic-manner genes (Fig. 2C; Table S2) were screened out based on the criterion (expression level: JFK + DDP > JFK or/and DDP, JFK + DDP < JFK or/and DDP). KEGG pathway analysis suggests that these synergistic-manner genes are enriched in MAPK signaling pathways, pathways in cancer, p53 signaling pathway, et al. (Fig. 2D). Bioinformatics analysis results also suggest that the synergistic pro-apoptotic effect induced by combined treatment of JFK and DDP involves those synergistically expressed genes (Table 3; Fig. S4).Based on these Bioinformatics analysis results, four upregulated synergistic genes, including AIFM2, APOL1, PTPN6, and CASP1, and four downregulated synergistic genes, including JUN, JUNB, MYC, and TCF7L2, were selected for measuring the transcriptional alternation in A549 cells after exposure to JFK and DDP simultaneously. RT-qPCR validation results indicate that nearly all tested genes present significant synergistic upregulation or downregulation after exposing A549 cells to JFK and DDP simultaneously (Fig. 2E). These results suggest modulation of transcriptional activity of the genes previously mentioned is potentially involved in the pro-apoptotic process in A549 cells.To investigate whether this phenomenon recurs in other human lung cancer cell lines, we performed a series of assays to examine the effect of combined treatment with JFK and DDP on NCI-H1975, NCI-H1650, and NCI-H2228 cell lines, respectively. We found that JFK plus DDP could deprive survival ability in a synergistic manner when treating on NCI-H1975, NCI-H1650, and NCI-H2228 cells (Fig. 3A, *P < 0.001). In addition, similar a phenomenon extends to cellular apoptosis, including both early and total apoptosis phases (Fig. 3B-D, *P < 0.001). Importantly, RT-qPCR results in NCI- H1975, NCI-H1650, and NCI-H2228 cells suggest that the majority of these genes expressed in a synergistic manner after being exposed to JFK and DDP simultaneously (Fig. 3E).