Inflammatory cytokine production in tumor cells ... - Semantic Scholar

RESEARCH ARTICLE

Inflammatory cytokine production in tumor cells upon chemotherapy drug exposure or upon selection for drug resistance Derek W. Edwardson1, Justin Boudreau2, Jonathan Mapletoft3, Carita Lanner1,2,3,4, A. Thomas Kovala1,2,3,4, Amadeo M. Parissenti1,2,3,4,5,6*

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1 Ph.D. Program in Biomolecular Science, Laurentian University, Sudbury, Ontario, Canada, 2 Department of Biology, Laurentian University, Sudbury, Ontario, Canada, 3 Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada, 4 Division of Medical Sciences, Northern Ontario School of Medicine, Sudbury, Ontario, Canada, 5 Health Sciences North Research Institute, Sudbury, Ontario, Canada, 6 Faculty of Medicine, Division of Oncology, University of Ottawa, Ottawa, Ontario, Canada * [email protected]

Abstract OPEN ACCESS Citation: Edwardson DW, Boudreau J, Mapletoft J, Lanner C, Kovala AT, Parissenti AM (2017) Inflammatory cytokine production in tumor cells upon chemotherapy drug exposure or upon selection for drug resistance. PLoS ONE 12(9): e0183662. https://doi.org/10.1371/journal. pone.0183662 Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute, UNITED STATES Received: February 26, 2017 Accepted: August 8, 2017 Published: September 15, 2017 Copyright: © 2017 Edwardson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All data may be obtained from Derek Edwardson, Ph.D. student with the Biomolecular Sciences Program at Laurentian University (derek.edwardson@gmail. com).

Tumor Necrosis Factor alpha (TNF-α) has been shown to be released by tumor cells in response to docetaxel, and lipopolysaccharides (LPS), the latter through activation of tolllike receptor 4 (TLR4). However, it is unclear whether the former involves TLR4 receptor activation through direct binding of the drug to TLR4 at the cell surface. The current study was intended to better understand drug-induced TNF-α production in tumor cells, whether from short-term drug exposure or in cells selected for drug resistance. ELISAs were employed to measure cytokine release from breast and ovarian tumor cells in response to several structurally distinct chemotherapy agents and/or TLR4 agonists or antagonists. Drug uptake and drug sensitivity studies were also performed. We observed that several drugs induced TNF-αrelease from multiple tumor cell lines. Docetaxel-induced cytokine production was distinct from that of LPS in both MyD88-positive (MCF-7) and MyD88-deficient (A2780) cells. The acquisition of docetaxel resistance was accompanied by increased constitutive production of TNF-αand CXCL1, which waned at higher levels of resistance. In docetaxel-resistant MCF-7 and A2780 cell lines, the production of TNF-α could not be significantly augmented by docetaxel without the inhibition of P-gp, a transporter protein that promotes drug efflux from tumor cells. Pretreatment of tumor cells with LPS sensitized MyD88positive cells (but not MyD88-deficient) to docetaxel cytotoxicity in both drug-naive and drug-resistant cells. Our findings suggest that taxane-induced inflammatory cytokine production from tumor cells depends on the duration of exposure, requires cellular drug-accumulation, and is distinct from the LPS response seen in breast tumor cells. Also, stimulation of the LPS-induced pathway may be an attractive target for treatment of drug-resistant disease.

Funding: The study was supported by a grant from the Northern Cancer Foundation of Sudbury, along with additional support for the purchase of reagents through a grant to Tom Kovala from the Northern Ontario School of Medicine, https://www.

PLOS ONE | https://doi.org/10.1371/journal.pone.0183662 September 15, 2017

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Cytokine expression in drug cytotoxicity and resistance

ncfsudbury.com/; http://www.nosm.ca/research/ default.aspx. Competing interests: Dr. Amadeo Parissenti and Dr. Jason Sprowl filed a patent application related to this investigation, but have since abandoned the patent application.

Introduction Breast cancer has a mortality rate second only to lung cancer [1,2]. Surgery is the primary treatment for most breast tumors in North America, followed by radiation and/or systemic adjuvant chemotherapy [3]. Neoadjuvant or ’preoperative’ chemotherapy is more common in other jurisdictions (namely Europe) and is often used worldwide to shrink tumors that are initially inoperable, permitting better surgical margins [4], as with locally advanced or inflammatory forms of the disease [5,6]. Despite continuous improvements in the treatment of solid tumors, response rates to chemotherapy are still relatively low and treatment side effects can be quite debilitating for patients. Treatment regimens for breast cancer in an adjuvant or neoadjuvant setting typically contain an anthracycline (doxorubicin or epirubicin) and a taxane (paclitaxel or docetaxel) [7]. The taxanes interact with β-tubulin, blocking the depolymerization of microtubules, and inhibiting cell division during mitosis [8,9]. As with breast cancer, treatment of ovarian cancer typically involves surgical removal of the tumor followed by adjuvant chemotherapy. Preoperative chemotherapy followed by interval debulking is also used in certain cases of advanced ovarian cancer [10]. In both instances, the chemotherapy drugs used typically involve the taxanes and a platinating agent [11]. Although the above chemotherapy agents have been shown to inhibit breast or ovarian tumor growth directly, in vitro, considerable evidence suggests that therapeutic benefit during treatment likely relies on an immunoadjuvant effect stemming from tumor cell death by different chemotherapy agents [12]. This effect involves the release of signaling molecules from tumor cells, serving as a trigger for a host immune response against the tumor. Various members of the family of damage-associated molecular patterns (DAMPs) have been shown to play a role as immunoadjuvants [13] emanating from dying tumor cells [14]; however, the full breadth of tumor-derived signals that are provoked during chemotherapy and that play a role in promoting a chemotherapy-induced tumor-targeted immune response is not clear. TNF-α is an inflammatory cytokine considered to be a master regulator of innate and adaptive immune responses [15,16] and a key player in many inflammatory disorders [17]. It has been shown to be released by and impart effects on a variety of cell types, including myeloid cells [18–20], endothelial [21], epithelial cells [22], as well as tumor cells. Recently, it has been shown in vitro that TNF-α is released by breast and ovarian tumor cells in response to taxane exposure [23]. The release of soluble factors, such as TNF-α, from tumor cells may be of importance in chemotherapy response, with and without the involvement of the host immune system. Cancer patients may not receive a benefit from chemotherapy due to innate resistance to chemotherapy drugs, involving pre-existing tumor characteristics, or due to acquired resistance, involving changes within the tumor or its microenvironment during treatment. The presence of a variety of factors including inflammatory cytokines [TNF-α, CXCL8 (interleukin-8), and CXCL1 (GRO-α)] have been implicated in mediating both innate and acquired resistance to taxanes and/or platinating agents in tumor cell lines [23–26]. Furthermore, the production of TNF-α by malignant cells in mice has been shown to affect tumor-associated myeloid cell activity, in turn affecting tumor growth [19]. TNF-α can also stimulate cell death pathways in tumors, as docetaxel-induced TNF-α production was shown to be cytotoxic in breast tumor cells via autocrine signaling [23]. It can also affect the tumor vasculature [27], which is an important element in the treatment of solid tumors. Poor treatment efficacy can be the result of inadequate drug exposure or penetration of the tumor, both of which can be due to a variety of factors including changes or anomalies in vascular architecture [28]. Chemotherapy-induced cytokine release has been reproducibly observed in mouse myeloid cells and it is thought to be mediated by activation of the pathogen recognition receptor known as toll-like receptor 4 (TLR4) [29,30]. However, the mechanism of chemotherapy-


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induced cytokine release in tumor cells is less understood. We hypothesize (a) that TNF-α release can be induced by a variety of chemotherapy agents, (b) that docetaxel-induced TNF-α release is an active cellular process, and (c) that the primary mechanism of docetaxel-induced TNF-α release is not attributable to direct ligand-binding of drug to TLR4 at the surface of human tumor cells. Multiple groups hold the view that taxanes activate TLR4 directly as a ligand in tumor cells [30–34] despite a lack of definitive evidence in tumor cells. We show evidence in human tumor cells that docetaxel-induced TNF-αrelease requires intracellular accumulation of drug. We also show that the molecular pathway involved in docetaxel-induced TNF-α release is distinct from that of lipopolysaccharides (LPS). Moreover, it does not require the presence of the TLR4 adaptor protein MyD88. We also show that the acquisition of resistance to docetaxel is accompanied by distinct changes in the expression of a variety of inflammatory cytokines, including TNF-α, CXCL1, and CXCL8. Finally, we demonstrate that stimulation of the TLR4 pathway in vitro can increase the sensitivity of tumor cells to the chemotherapy agent docetaxel, thus identifying a possible approach to increase the efficacy of this drug in docetaxel-resistant disease.

Methods Cell culture and cell lines MCF-7 cells were obtained from the American Tissue Culture Collection, while A2780 cells were from the European Collection of Cell Cultures. Cells were cultured in DMEM and RPMI media using Sarstedt and Corning T75 tissue culture flasks for MCF-7 and A2780 cells, respectively, in a 5% CO2 atmosphere at 37˚C. Culture media was supplemented with 10% FBS (v/v) and 1% Penicillin and Streptomycin (v/v). At confluence, cells were washed with PBS, treated with 0.25% trypsin for resuspension, after which 1 ml of complete media was added to inhibit trypsin activity. Cell suspensions were harvested by centrifugation at 650 x g for 7 minutes and resuspended in medium for counting. Docetaxel-resistant variants were generated from MCF7 cells and A2780 cells as previously described by Guo et al. [35] and Armstrong et al. [36], respectively. MCF-7TXT7, MCF-7TXT8, MCF-7TXT9, MCF-7TXT10, MCF-7TXT11, and MCF-7TXT12 cells are MCF-7 cell lines selected for survival in progressively increasing concentrations (doses) of docetaxel [also known as Taxotere1 (TXT)]. These doses (7 through 12) are 0.37, 1.1, 3.3, 5, 15, and 45 nM docetaxel, respectively. Likewise, A2780DXL10, A2780DXL11, and A2780DXL12 cells represent a series of A2780 cell lines selected for survival in up to 1.97 nM (dose 10), 2.96 nM (dose 11), and 8.88 nM (dose 12) docetaxel, respectively. During the above selections for docetaxel resistance, equivalent cultures of MCF-7 and A2780 cells were passaged in the absence of drug to control for differences in cell properties and behavior due to increased passage number. One of these “co-cultured control” cell lines at selection dose 10 for the MCF-7 cell line and at selection dose 12 for the A2780 cell line (MCF-7CC10 and A2780CC12 cells, respectively) served as the drug-sensitive control cell line in experiments.

ELISA (enzyme-linked immunosorbent assay) All ELISAs were completed using kits from R&D systems (Minneapolis, MN, USA), following the manufacturer’s experimental protocol. Plates pre-coated with monoclonal antibody were purchased for the detection of the cytokines TNF-α, CXCL8, and CXCL1 (catalog numbers DTA00C, D8000C and DGR00, respectively). Equal numbers of cells were transferred to 10 cm culture plates and allowed to adhere overnight. After treatment with docetaxel in the absence or presence of various additional agents for up to 96 hours, the culture media were collected and subjected to centrifugation at 875 x g to remove cell debris. The media supernatants were then transferred to a 50 ml Amicon tube equipped with a 3 kDa molecular-weight-


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cut-off filter (EMD Millipore, Etobicoke, ON; cat no. UFC900324) and subjected to centrifugation at 3273 x g. The volumes of media supernatants were measured and the samples stored at -80˚C. ELISA standards were prepared by serial dilution of a reconstituted lyophilized stock solution. After 2 hours of incubation at room temperature, the wells were rinsed with wash buffer. A horseradish peroxidase (HRP)-conjugated secondary antibody was then added to each well and the plate incubated for 1 hour at room temperature, followed by a rinse with wash buffer and addition of HRP-sensitive colorimetric substrate for 20 minutes. The absorbance of the samples at 450 nm and 540 nm was measured using a Synergy H4 Hybrid spectrophotometer from BioTek1 and duplicate measurements were averaged for both samples and standards. After subtracting the absorbance at 540 nm from the absorbance at 450 nm absorbance values were likewise corrected for background absorbance of standard diluent (for standards) or concentrated tissue culture medium (for samples).

Cell counting and trypan blue staining Following trypsinization and staining with 0.066% trypan blue (v/v), cells were counted in a hemocytometer (Improved Neubauer, Hausser Scientific) using a light microscope (Leica 10x/0.22) with phase contrast filter. The average number of trypan-positive and trypan-negative cells within a 4x4 grid of 4 different fields was determined.

Reverse-transcription of mRNAs and quantitative PCR (polymerase chain reaction) Two million cells were transferred to 10 cm tissue culture dishes and grown for 30, 36, and 42 hours. RNA was isolated using a Quiagen RNeasy extraction kit (Qiagen, Inc., Toronto, ON). The cells were washed with PBS, and treated with 350 μl of RLT buffer containing 3.5 μl of βmercaptoethanol. Cells were harvested using a cell-scraper, and the lysate was subjected to 4–5 passes through a 16-gauge needle. After transfer of the lysate to a microfuge tube, an equal volume of 70% ethanol was added, and after mixing, the mixture was transferred to a Qiagen mini-column and RNA purified as described in the manufacturer’s protocol. The concentration of purified RNA was determined by measuring the absorbance at 260 nm in a NanoDrop 2000C spectrophotometer (Thermo Scientific), with a baseline correction for absorbance at 340 nm. Two μg of purified RNA was diluted to generate a mixture of 15 μl, containing 2 μl of 10x DNase buffer, and 2 ul of DNase (1 unit/μl). After a 15 minute incubation at room temperature, 2 μl of 25 mM ethylenediaminetetraacetic acid (EDTA), 20 μl of T20 primers (20 ng/μl) and 16 μl of dNTP’s (10 mM) were added. Samples were incubated for 5 minutes at 65˚C, after which 16 μl of 5x first-strand buffer and 8 μl of dithiothreitol (DTT) were added, with a subsequent incubation period of 2 minutes at 37˚C. Finally, 200 U of Moloney-murine leukemia virus (M-MLV) reverse-transcriptase was added and the mixture left for 2 hours at 37˚C to allow for reverse-transcription of mRNA to cDNA. Samples were then heated for 5 minutes at 95 oC to inactivate the transcriptase. Quantitative real-time PCR reactions were performed in a 96-well plate using cDNA preparations. mRNA standards were generated using cDNA from an untreated sample originating from the same cell line as the other samples, to generate a standard curve. Five or more 2-fold serial dilutions were made starting with an initial 4-fold dilution in RNase-free water. Samples were prepared beginning with a 16-fold dilution (3 μl of sample added to 45 μl of RNase-free water). The primer pair for detection of TNF-α transcripts was 5'-CCT GCC CCA ATC CCT TTA TT-3' (forward) and 5'-CCC TAA GCC CCC AAT TCT CT-3' (reverse). A primer pair with sequences 5'-TCC ATC ATC CGC AAT GTA AAA-3' (forward) and 5'GCT TCT CGC TCT GAC TCC AAA-3' (reverse) was also used to quantify expression of the S28 reference


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gene. All primers were purchased from Integrated DNA Technologies (Skokie, IL, United States). Triplicate samples were prepared for qPCR with 5 μl per well of cDNA standards, including a notemplate control containing only RNase-free water, or 5 μl per well of cDNA for unknown samples, to which was added 7.5 μl of 2 mM pooled forward and reverse primers and 12.5 μl per well of 1x SYBR Green. The plate was centrifuged for 1 minute at 650 x g in order to position all reagents at the bottom of each well. qPCR involved a hot start for 5 minutes at 95˚C, followed by 40 cycles consisting of: 30 seconds at 95˚C, 30 seconds at 53˚C, and 1 minute at 72˚C.

Flow cytometry Cells (2 x 105 per well) were allowed to adhere overnight in 6-well plates. The cells were rinsed with PBS, resuspended with 500 μl of 0.25% trypsin, transferred to 1 ml microfuge tubes, combined with 500 μl of PBS, and centrifuged for 3 minutes at 300 x g. The pelleted cells were resuspended in a mixture of 20 μl of phycoerythrin-conjugated monoclonal antibody, specific for P-glycoprotein (P-gp), protected from light and left at room temperature for 30 minutes. Cells were centrifuged for 3 minutes at 300 x g, washed with PBS, resuspended in PBS and then analyzed with an FC500 flow cytometer (Beckman Coulter, Mississauga, ON) set to measure fluorescence using the FL2 filter for 10,000 events per sample with no gating. Mean fluorescence Intensity (MFI) values for the mouse anti-human P-gp antibody or for an IgG2β isotype control (BD Bioscience, catalog 557003 and 555743, respectively) were recorded and the MFI for the isotype control subtracted from the MFI for the P-gp-specific antibody to correct for non-specific binding.

Drug uptake measurements Cells (2x105) were plated in 6-well plates and left to adhere overnight in the appropriate medium. Cells were then incubated with either 2.5 nM tritium-labeled docetaxel (3H-TXT from American Radiolabeled Chemical, St. Louis, MO) or 100 nM Tariquidar (Med Chem Express, Monmouth Junction, NJ) or a combination thereof, with 5% CO2 at 37˚C. After 12 hours, the media were removed, while the adhered cells were rinsed with 1 ml PBS, treated with 0.25% trypsin, and then placed in 5 ml of scintillation fluid. The radioactivity associated with the cells was then quantified using a Beckman LS 6000 IC scintillation counter.

Cell protein extraction Two million A2780 and MCF-7 cells were seeded onto 10 cm tissue culture plates and cultured for 24 and 48 hours, respectively. Protein extracts were prepared from these cells by lysis in 500 μl of chilled RIPA buffer [1% NP-40 (Sigma, St. Louis, MO), 0.5% sodium deoxycholic acid (Sigma, Oakville, ON) and 1% SDS (BioRad, Mississauga, ON) in PBS] supplemented with 2 mM sodium orthovanadate (Sigma) and 1X protease inhibitor cocktail (Roche, Mississauga, ON). Cell lysates were passed through a 21-gauge needle 5 times, incubated for 30 minutes on ice, and then centrifuged at 15,000 x g for 30 min. The supernatants were divided into aliquots that were stored at -80˚C. The protein concentration of extracts was quantified using the Pierce BCA protein assay kit (Thermo-Fisher, Mississauga, ON). Alternatively, cells were extracted in 5X SDS-PAGE sample buffer to ensure complete dissolution of membranes for enhanced isolation of membrane-associated proteins.

Immunoblotting experiments Lysates (36 μg of protein) were loaded onto a 10% polyacrylamide gel for electrophoresis, transferred to a nitrocellulose membrane using a BioRad Trans-blot1 SD Semi-Dry Transfer


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Cell for 1hr at 12V. The membrane was blocked in 5% skim milk in TBST [0.24% Trizma1 Base (Sigma), 0.8% NaCl (Fisher), 0.1% Tween201 (Sigma) at pH 7.6] for 1 hour before incubation overnight at 4˚C with a human TLR4 antibody (1:250, Santa Cruz, Dallas, TX), human MyD88 antibody (1:1000, Cell Signaling, Danvers, MA), human TRIF antibody (1:700, Cell Signaling, Danvers, MA) or a GAPDH antibody (1:10,000, Santa Cruz). All primary antibodies were diluted in TBST supplemented with 5% BSA (Sigma). Membranes were washed with TBST 3 times for 5 minutes. TLR4 and MyD88 levels were detected using an HRP-conjugated goat anti-rabbit secondary antibody (1:10,000, Santa Cruz). The membranes were washed before incubation with ECL reagent (Santa Cruz) and exposure on CURIX ortho HTL-Plus film (AGFA Healthcare, Waterloo, ON). Densitometry was performed using the FluorChem FC3 apparatus and AlphaEaseFC 4.0 software.

Clonogenic assays Cells were added to 6-well plates (3x105 cells) with 2 ml of media per well and left to adhere overnight. Each well received one of twelve different concentrations of docetaxel (including a ‘no drug’ control) and the cells incubated for 24 hours. After 24 hours the medium in each well was collected, while the adherent cells were resuspended using 0.25% trypsin. The floating and adherent cells from each well were combined, centrifuged at 650 x g for 7 minutes, and resuspended in 300 μl of culture media. The cells were then added to 2.7 ml of methylcellulose (with 25% FBS v/v), and mixed thoroughly by vortexing. After incubating an hour to allow bubbles to escape, 1.2 ml of each cell suspension were transferred to a 6-well plate. The plates were then incubated for two weeks and photographs of 12 independently chosen fields were taken using a Leica light microscope with a Leica 4x/.10 objective lens. Viable colonies, defined as being greater than or equal to 4 mm (MCF-7) or 3 mm (A2780) in two perpendicular directions (as measured on a PowerPoint slide and printed 4 slides per 8’x11’ page), were counted. The average colony count was then computed and divided by the average colony count for the untreated control to obtain the survival fraction. Survival fractions were then plotted against drug dose and a sigmoidal dose-response (survival) curve was fit to 12 data points using GraphPad Prism software. The concentration that inhibited colony formation by 50% (IC50) was determined for each dataset at the inflection point of the sigmoidal curve. Clonogenic assays using Tariquidar involved 24-hour co-administration of Tariquidar and docetaxel together, whereas clonogenic assays with LPS involved a 48-hour pretreatment with LPS followed by a 24-hour docetaxel treatment. Tariquidar clonogenics used 3 x 105 MCF-7 cells and 2 x 105 A2780 cells, whereas LPS clonogenics used 2 x 105 and 1 x 105 cells for MCF-7 and A2780 cells, respectively.

Statistical analyses Statistical analyses are specified in figure legends for each experiment. They involved comparison of the mean values from data obtained from at least three replicates (separate cell culture dishes). Two-tailed T-tests were performed when comparing data, from cell populations, with one independent variable (ie. drug concentration or time after treatment). 2-way Analysis of Variance (ANOVA) was performed for experiments comparing data from cell populations with two independent variables (ie. both drug concentration and time).

Results Characterization of docetaxel-induced TNF-α release Assessing the effects of drug concentration and exposure time on TNF-α release. The earliest reports of taxane-induced TNF-α production involved the treatment of mouse


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Fig 1. Docetaxel-induced TNF-α levels in media of breast and ovarian tumor cells are dose- and time-dependent. A) and B) Effects of treatment time on TNF-α release by 2.5 nM docetaxel from MCF-7 and A2780 cells; C) and D) Effects of docetaxel concentration on TNF-α release in breast and ovarian cancer cell lines over 72 hours. The data represents the mean of three replicates (+ SEM). The significance of differences in secreted TNF-α levels between treated and untreated (NT) cells was assessed using a two-tailed T-test;*** p