Augmented expression of cardiac ankyrin repeat protein is induced by pemetrexed and a possible marker for the pemetrexed resistance in mesothelioma cells

Human mesothelioma cells, NCI-H28, NCI-H226, MSTO-211H and NCI-H2452, and immortalized Met-5A cells of mesothelium origin were purchased from American Type Culture Collection (Manassas, VA, USA), and mesothelioma, EHMES-1 and JMN-1B cells, were provided by Dr. Hironobu Hamada (Hiroshima University, Japan) [13]. PEM-resistant H28-PEM, H226-PEM, 211H-PEM, and H2452-PEM cells were previously established by a stepwise increase of PEM (Eli Lilly, Indianapolis, IN, USA) [12]. Cells were cultured with in RPMI-1640 medium supplemented with 10% fetal calf serum, and confirmed to be negative for mycoplasma. The genotype of p53 was wild-type in NCI-H28, NCI-H226, MSTO-211H and NCI-H2452 cells but p53 protein of NCI-H2452 cells was truncated [14]. In contrast, the genotype of EHMES-1 and JMN-1B cells was mutated. A769662 (Abcam, Cambridge, UK) and nutlin-3a (Selleck, Houston, TX, USA) were used to stimulate endogenous the AMPK and the p53 pathways, respectively.

An aliquot of total RNA was labeled with a fluorescence dye and hybridized with a whole human genome array (44Kx4 ver 2.0, Agilent Technologies, Santa Clare, CA, USA). Expression of respective genes and clustering of the gene expression was analyzed with GeneSpring GX11.5 (Agilent).

Cells were transfected with small interfering RNA (si-RNA) duplex targeting cardiac ankyrin repeat protein (CARP) (si-RNA-s502326, s502327, s502328) (Thermo Fisher Scientific, Fremont, CA, USA), insulin-like growth factor binding protein-3 (IGFBP3) (si-RNA-s7227, s7228, s7229) (Thermo Fisher Scientific), or nonspecific si-RNA as a control (Thermo Fisher Scientific) using Lipofectamine RNAiMAX according to the manufacturer’s protocol (Thermo Fisher Scientific).

Total RNAs were isolated with TRIzol reagent (Thermo Fisher Scientific) from cells transfected with siRNAs for IGFBP3. First-strand cDNA was synthesized from the RNA preparations using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and amplification of equal amounts of the cDNA was performed with the following primers and conditions: for the IGFBP3 gene, 5′-GACAGAATATGGTCCCTGCCG-3′ (forward) and 5′-TTGGAAGGGCGACACTGCT-3′ (reverse), and 15 s at 95 °C for denature/45 s at 60 °C for annealing/26 cycles; for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 5′-TCCACCACCCTGTTGCTGTA-3′ (reverse), and 15 s at 94 °C/15 s at 60 °C/25 cycles.

Cells were seeded into 96-well plates (2.8 × 10³ cells per well), treated with si-RNA or human recombinant IGFBP3 (Wako, Osaka, Japan) for 24 h and then incubated with PEM for 72 h. Cell viabilities were assessed with a WST-8 kit (Dojindo, Kumamoto, Japan) and the relative viability was calculated based on the absorbance at 450 nm without any treatments (WST assay). GraphPad Grism ver 6.0 (GraphPad Grism Software, San Diego, CA, USA) was used to calculate 50 or 75% inhibitory concentrations (IC₅₀ and IC₇₅).

Cell lysate was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The protein was transferred to a nitrocellulose membrane and was hybridized with antibody against integrin β-3 (Catalog Number: #13166), AMPK (#2532), phosphorylated AMPK (Thr 172) (#2535), phosphorylated p53 (Ser 15) (#9284) (Cell Signaling, Danvers, MA, USA), plasminogen activator inhibitor (#ab20562), a disintegrin and metalloproteinase with thrombospondin motifs 5 (#ab41037) (Abcam), IGFBP3 (#sc-9028), CARP (sc-30181), β-2 adrenergic receptor (#sc-569) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated H2AX (Ser 139) (#613401) (BioLegend, San Diego, CA, USA), p53 (Ab-6, Clone DO-1) and tubulin-α (Clone DM1A) (Thermo Fisher Scientific) as a control followed by an appropriate second antibody. The membranes were developed with the ECL system (GE Healthcare, Buckinghamshire, UK).

IGFBP3 concentrations in culture supernatants and cell lysate were measured with a human IGFBP3 ELISA kit (R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions. Optical density was measured based on the absorbance at 450 nm using a micro-plate reader.

We established PEM-resistant cells, H28-PEM, H226-PEM, 211H-PEM and H2452-PEM, from respective parent cells, NCI-H28, NCI-H226, MSTO-211H and NCI-H2452, and showed the resistance with a colony-forming assay [12]. We then demonstrated the resistance with the WST assay by measuring relative cell viability after an exposure to various PEM concentrations (Fig. 1). We confirmed that all kinds of the PEM-resistant cells were less sensitive to PEM than their parent cells. NCI-H28, NCI-H229 and MSTO-211H cells showed similar PEM sensitivity but NCI-H2452 cells were relatively resistant to PEM.

We previously showed that both 211H-PEM and H2452-PEM cells elevated TS and GARFT mRNA expression in comparison with respective parent cells, whereas the expression levels of H28-PEM and H226-PEM cells were not elevated or rather lower than those of their parent cells [12]. The DHFR transcripts in H28-PEM and H226-PEM cells also decreased compared with the parent cells [12]. We thereby selected two kinds of paired cells, NCI-H28 and H28-PEM, and NCI-H226 and H226-PEM cells, for further analyses to select the genes which elevated the expression greater in PEM-resistant than in the parent cells. We first conducted a microarray analysis which compared gene expression profiles of parent cells with that of PEM-resistant cells in the four kinds of mesothelioma cells (Additional file 1: Fig. S1). We included CDDP-resistant mesothelioma cells in the analyses, which were also established with the same method as the PEM-resistant cells [12]. The analysis detected 21,964-24,509 spots in these parent cells and similar spot numbers were also found in respective CDDP- and PEM-resistant cells. A hierarchical clustering analysis with 19,307 spots revealed that differential expression profiles of respective parent cells were distinctive among these parent cells more than those between individual parent cells and the respective CDDP- or PEM-resistant cells. We therefore screened genes of which the expression were up-regulated in PEM-resistant cells 5 times greater than that in the parent cells, and identified six genes from the two kinds paired cells (Table 1).

We examined expression levels of the six up-regulated genes in four paired cells with Western blot analysis (Fig. 2). The analysis showed that CARP expression was augmented in PEM-resistant cells except H2452-PEM cells. IGFBP3 expression showed multiple bands and the intensity in total seemed to increase in all the 4 PEM-resistant cells. The band of the highest molecular weight (50 kDa) however turned out to be a non-specific signal based on experiments with the si-RNA for IGFBP3 and ELISA (see below). The rest of IGFBP3 bands represented several isoforms migrated at molecular weights between 40 and 44 kDa. The IGFBP3 expression in H226-PEM cells could be greater than that in NCI-H226 cells, whereas the expression in H28-PEM cells, showing only a non-specific band at 50 kDa, rather decreased in comparison with that in the parent cells expressing the multiple isoforms. The IGFBP3 level in 211H-PEM and H2452-PEM cells was marginally greater than that of the respective parent cells. In contrast, expression of other molecules, PAI1, ADRB2, ADAMTS5 and ITGB3, was not different between the PEM-resistant and the parent cells. We selected CARP and IGFBP3 based on the differential expression levels and further examined the functions in PEM-resistance. We currently do not know a reason of the discrepant levels between mRNA and protein in respective paired cells.

We examined a possible role of CARP in the PEM resistance by down-regulating the expression with si-RNA. We firstly investigated transfection efficiency with different doses of Alexa Fluor red fluorescent control in H28-PEM cells (Additional file 2: Table S1). The fluorescence values showed that 10 nM si-RNA was enough to obtain sufficient efficacy. H28-PEM cells were then treated with three kinds of si-RNA for CARP (s502326, s502327 and s502328) and each kind of si-RNA down-regulated the CARP expression (Fig. 3a). The cells, irrespective of the si-RNA, did not show any changes in their sensitivity to PEM (Fig. 3b). We also tested 221H-PEM cells, expressing CARP greater than parent MSTO-211H cells, and found that the si-RNA treatments did not enhance the PEM sensitivity (Additional file 3: Fig. S2). These results suggested that elevated CARP expression did not contribute to the PEM resistance.

We measured the production of IGFPB3 in culture supernatants and cell lysate with an ELISA kit (Table 2). H226-PEM cells produced IGFBP3 greater than the parent cells in culture supernatants and cell lysate, but H28-PEM cells rather produced less amounts of IGFBP3 compared with the parent cells. These ELISA data were concordant with the expression levels detected with Western blot analysis. We further examined a possible role of IGFBP3 in the drug resistance since IGFBP3 influenced IGF-mediated signaling which was linked with the drug insensitivity [15]. We transfected three kinds of si-RNA for IGFBP3 (s7227, s7228, and s7229) into H226-PEM cells and examined the expression levels with their parent cells with ELISA (Additional file 4: Fig. S3) and Western blot analysis (Fig. 4a). ELISA data showed decreased production of IGFBP3 and Western blot data indicated that the band corresponding the highest molecular weight remained unchanged in the intensity, but lower bands below the highest became significantly weak. We also examined various doses of si-RNA and confirmed down-regulated IGFBP3 transcripts (Fig. 4b). The si-RNA treatments showed that intensity of the lower bands decreased in a dose-dependent manner (Fig. 4c). These si-RNA experiments confirmed that the lower molecular bands but not the highest band corresponded to IGFBP3. We then tested the PEM sensitivity of H226-PEM cells treated with the si-RNA and showed that knocking down of IGFBP3 did not improve susceptibility to PEM (Fig. 4d). We also examined whether recombinant IGFBP3 decreased PEM sensitivity to NCI-H226 cells but the exogenous IGFBP3 did not affect the sensitivity in a dose-dependent manner (Additional file 5: Fig. S4). These data collectively showed that IGFBP3 was not involved in susceptibility to PEM.

We examined possible augmentation of CARP and IGFBP3 expression with PEM treatments. We treated paired cells with PEM and investigated the expression with Western blot analysis (Fig. 5). Both NCI-H28 and H28-PEM cells augmented CARP expression, but up-regulation of the IGFBP3 was minimal. NCI-H226 cells and less significantly H226-PEM cells showed increased CARP expression with PEM treatments, but the IGFBP3 expression was minimally or scarcely changed. These data therefore showed that PEM treatments up-regulated CARP but not IGFBP3 expression.

We investigated a mechanism of the constitutive increase of CARP expression by treating cells with PEM (Fig. 5). We firstly examined p53 expression after PEM treatments in NCI-H28 and NCI-H226 cells, both of which had the wild-type p53 genotype, and in the PEM-resistant cells. The PEM treatments induced phosphorylation of p53 at serine 15 in NCI-H28, H28-PEM and NCI-226H cells, and consequently the p53 levels were up-regulated in these cells. In contrast, H228-PEM cells did not show the augmented p53 expression or phosphorylated p53, which could be attributable to less sensitivity of the cells to PME-mediated cytotoxicity. We then examined induction of phosphorylated H2AX, a DNA damage marker, and found that the enhanced level in H228-PEM cells was less than that in the other cells which showed the increased expression in a time- and a dose-dependent manner. These data suggested that the up-regulated level of CARP was associated with a degree of DNA damages followed by activation of the p53 pathways. We therefore examined whether the CARP up-regulation was linked with the p53 genotype. We used mesothelioma cells with mutated p53 genotype, EHMES-1 and JMN-1B, and mesothelium-derived immortalized Met-5A cells that expressed the SV40 T antigen (Fig. 6). PEM-treated EHMES-1 cells augmented CARP expression levels, but JMN-1B cell and more remarkably Met-5A cells decreased the expression, whereas phosphorylated H2AX levels increased in all the cells upon PEM treatments. These data thereby indicated that PEM-mediated CARP augmentation was not directly dependent on DNA damages. We further examined whether augmentation of endogenous p53 without DNA damages contributed to increased CARP expression. Nutlin-3a inhibited interaction between p53 and MDM2 molecules which mediated ubiquitination of p53 and subsequent p53 degradation [16]. Nutlin-3a-treated cells with the wild-type p53 therefore increased p53 levels due to inhibiting the p53 degradation processes (Fig. 7). NCI-H28 and H28-PEM cells treated with nutlin-3a increased CARP expression levels, whereas treated NCI-H226 and H226-PEM cells rather decreased the CARP levels except a temporally increased NCI-H226 sample treated at 20 μM for 48 h. Phosphorylated levels of H2AX were dependent on a cell type and a treated dose. Cells originated from NCI-H28 cells increased endogenous p53 without increased H2AX phosphorylation but those from NCI-H226 cells augmented the phosphorylation at 50 μM nutlin-3a treatments. Nutlin-3a was not essentially genotoxic but treatments at a high concentration induced DNA damages in cells of NCI-H226 origin. These data collectively indicated that a way of CARP induction was subjected to cell type difference. Activation of the p53 pathways in NCI-H28 cells was associated with the increased CARP expression but DNA damages was irrelevant. In contrast, neither p53 up-regulation nor DNA damages were linked in cells derived from NCI-H226 cells.

We also examined the effects of PEM on AMPK expression and the phosphorylation (Fig. 5). All the cells including H226-PEM cells increased phosphorylation of AMPK but the AMPK levels remained unchanged, which indicated that PEM stimulated the AMPK signal pathway. These data raised possibility that PEM-mediated augmentation of CARP expression was attributable to activation of the AMPK pathway. We then examined a role of the AMPK activation with an AMPK stimulating agent, A769662 (Fig. 8). NCI-H28 and NCI-H226 cells treated with A769662 increased phosphorylated AMPK, but the CARP expression was not induced or rather decreased. IGFBP3 was not induced by A769662 either. These data showed that activation of AMPK pathway was not involved in the PEM-mediated CARP augmentation.