A Nonsteroidal Anti-inflammatory Drug, Zaltoprofen, Inhibits the Growth of Extraskeletal Chondrosarcoma Cells by Inducing PPARγ, p21, p27, and p53

doi:10.21203/rs.3.rs-469815/v1

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A Nonsteroidal Anti-inflammatory Drug, Zaltoprofen, Inhibits the Growth of Extraskeletal Chondrosarcoma Cells by Inducing PPARγ, p21, p27, and p53

https://doi.org/10.21203/rs.3.rs-469815/v1

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There is no effective treatment except surgical resection for extraskeletal myxoid chondrosarcoma (EMC). Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor and a master transcription factor of adipogenesis-related genes and has been reported as an antitumor target for chondrosarcomas. Herein, we show that a nonsteroidal anti-inflammatory drug, zaltoprofen, induces the expression of PPARγ at mRNA and protein levels, following the induction of PPARγ-activating factors, such as Krox20, C/EBPβ, and C/EBPα, in human extraskeletal chondrosarcoma cells, H-EMC-SS. Zaltoprofen inhibited the proliferation of these cells in vitro. Upregulation of cell cycle checkpoint proteins, p21, p27, and p53, was observed upon treatment of cells with zaltoprofen, which probably resulted in the inhibition of proliferation of H-EMC-SS cells. Zaltoprofen treatment induced tumor necrosis, which could be due to its antiproliferative properties, and was well tolerated in a mouse model of EMC. Our results provide mechanistic insights into the therapeutic effect of zaltoprofen that should promote further studies on the rational use of this drug for effective treatment of EMC.

Cancer Biology

Health Economics & Outcomes Research

surgical resection

A Nonsteroidal Anti-inflammatory Drug

Zaltoprofen

Extraskeletal Chondrosarcoma Cells

Extraskeletal myxoid chondrosarcoma (EMC) is a soft tissue sarcoma that arises at various sites, including the extremities¹. Radiation and chemotherapy being ineffective, wide resection is the only radical treatment available for EMC ¹. Although considered a slow-growing neoplasm, the recurrence and metastatic rates of EMC after radical resection (with values up to 50% for each) are higher than those for soft-tissue sarcoma ^1,2. Therefore, development of novel treatment strategies is crucial for long-term survival of patients with EMC.

We recently reported that peroxisome proliferator-activated receptor gamma (PPARγ), a ligand-activated nuclear receptor that controls the differentiation of adipocytes in normal tissues, could be a therapeutic target for inhibiting cell invasion and matrix metalloproteinase-2 (MMP2) expression in chondrosarcoma cells ³. In addition, we demonstrated that zaltoprofen, a nonsteroidal anti-inflammatory drug (NSAID), could effectively induce and activate PPARγ and elicits antitumor effects in chondrosarcoma cells ³.

To further understand the therapeutic potential of PPARγ-targeted treatment using zaltoprofen, in the present study, we investigated the mechanism of zaltoprofen-mediated induction of PPARγ and its anti-proliferative effect in vitro and in vivo on human EMC cells.

Reagents.

Zaltoprofen was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and was dissolved in 10% dimethyl sulfoxide (DMSO) for in vitro experiments or in 0.2% carboxymethylcellulose sodium salt (CMC-Na) for in vivo experiments.

Cell culture.

The human EMC cell line, H-EMC-SS, was cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with fetal bovine serum (10%), penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37℃.

RNA interference.

PPARγ knockdown was conducted using human PPARγ short hairpin RNA (shRNA) (SureSilencing shRNA plasmid, Qiagen, Valencia, CA, USA). Attractene transfection reagent (Qiagen) was used to transfect the PPARγ shRNA (shPPARγ) plasmid or nontargeting shRNA (shNT) control plasmid in H-EMC-SS cells. Neomycin (G418 solution, Roche Diagnostics K.K., Tokyo, Japan) was used for the selection of shRNA-transfected cells, and the knockdown efficiency was confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Thus, PPARγ knockdown cell line (H-EMC-SS^shPPARγ) and nontarget control cell line (H-EMC-SS^shNT) were established. The shRNA insert sequences were as follows: PPARγ shRNA, 5′-CCACGAGATCATTACACAAT-3′; nontargeting shRNA, 5′-GGAATCTCATTCGATGCATAC-3′.

RT-qPCR.

After treatment with zaltoprofen for 24 h, H-EMC-SS cells were lysed with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and total RNA was extracted. The RNA samples (100 ng/sample) were quantified using a spectrophotometer (NanoDrop Lite, Thermo Scientific K.K., Tokyo, Japan), and reverse-transcribed to cDNA on a T100 Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using an AffinityScript cDNA Synthesis Kit (Agilent Technologies Inc., Santa Clara, CA, USA). RT-qPCR was performed with 10 ng cDNA per well on a StepOne™ Real-time PCR System (Applied Biosystems, Foster City, CA, USA) using the QuantiTect SYBR Green PCR Kit (Qiagen). The primers for PPARγ, CCAAT/enhancer binding protein (C/EBP)α, C/EBPβ, C/EBP δ, and　Krox20 were purchased from Qiagen (QuantiTect Primer Assay, Hs_PPARG_1_SG, Hs_CEBPA_1_SG, Hs_CEBPB_1_SG, Hs_CEBPD_1_SG, and Hs_EGR2_1_SG). The sequences of primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: forward, 5′-TGCACCACCAACTGCTTAGC-3′; reverse, 5′-GGCATGGACTGTGGTCATGAG-3′.

Western blot analysis.

After treatment with various concentrations of zaltoprofen for 24 h, H-EMC-SS cells were lysed in RIPA buffer (1% sodium deoxycholate, 150 mM NaCl, 50 mM sodium fluoride, 100 μM sodium orthovanadate, 0.1% sodium dodecyl sulfate [SDS], 1% Triton X-100, 10 mM Tris, pH 7.2) containing a protease inhibitor cocktail (Sigma-Aldrich Japan, Tokyo, Japan). Total protein (100 µg/sample) was boiled (2 min at 95 °C) and resolved by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) on an e-PAGEL (ATTO Co., Tokyo, Japan), and transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore Corp., Bedford, Germany). After overnight incubation in blocking solution (Odyssey Blocking Buffer PBS, LI-COR Corp., Lincoln, NE, USA), the membranes were incubated with the following antibodies at 4 °C for 60 min: anti-PPARγ antibody (Santa Cruz), anti-p21 (Cell Signaling Technology, Danvers, MA, USA), anti-p27 (Santa Cruz), anti-p53 (Santa Cruz), and anti-GAPDH (Cell Signaling Technology). The membranes were washed three times with phosphate-buffered saline containing 10% Tween 20 (T-PBS) and incubated with the secondary antibody (IRDye 680RD goat anti-IgG secondary antibody, LI-COR) for 30 min. After washing three times with T-PBS, the bands were visualized and scanned with an Odyssey Infrared Imaging system (LI-COR).

Water-soluble tetrazolium salt-8 (WST-8) assay.

The growth of cells at designated time points after zaltoprofen treatment was determined using a Cell Counting Kit-8 (Dojindo Laboratory, Kumamoto, Japan). After H-EMC-SS cells (5 × 10³ cells/well) were treated with the indicated concentrations of zaltoprofen, the absorbance at 450 nm was measured using an iMark Microplate Absorbance Reader (Bio-Rad Laboratories).

5-ethynyl-2′-deoxyuridine (EdU) proliferation assay.

After incubating the H-EMC-SS^shPPARγ and H-EMC-SS^shNT cells on slide chambers for 24 h, they were further incubated with 1% DMSO or 400 µmol/L zaltoprofen for 24 h. The cells were then treated with 10 mmol/L EdU stock solution (Click-iT^® EdU Alexa Fluor^® 555 Imaging Kit, Invitrogen) for 60 min, fixed with 4% paraformaldehyde (Wako), washed twice with 3% bovine serum albumin (BSA), permeabilized with 0.5% Triton X-100, incubated with the Click-iT reaction buffer (Click-iT^® EdU Alexa Fluor^® 555 Imaging Kit) for 30 min, and washed once with BSA. The nuclei of the cells were stained with Hoechst 33342 (NucBlue Live ReadyProbes Reagent, Invitrogen). Cells positive for EdU-stained nuclei were visualized with a fluorescence microscope (BZ-9000, Keyence Co., Osaka, Japan). EdU-positive and Hoechst 33342-positive cells were counted using the ImageJ software (National Institute of Health, USA).

Animal experiments.

Four-week-old BALB/c female nude mice (Charles River Laboratories Japan Inc., Kanagawa, Japan) were adapted to the conditions for a week before the experiment.

H-EMC-SS cells suspended in Matrigel (BD Japan, Tokyo, Japan) (4 × 10⁶/100 µL) were subcutaneously implanted on the flanks of mice. The mice were randomly divided into zaltoprofen (30 mg/kg, oral gavage, twice a day, 42 days)-treated (n = 6) and vehicle (0.2% CMC-Na, 10 mL/kg, oral gavage twice a day for 42 days)-treated (n = 6) groups. Tumor diameter and body weight of mice were measured once a week using digital calipers and digital scales, respectively. Tumor volume (mm³) was calculated using the following formula: Tumor volume (mm³) = largest diameter (mm) × smallest diameter (mm) × smallest diameter (mm) × 0.5.

After 42 days of treatment, the mice were humanely sacrificed and scanned by computed tomography (CT), and tumors were resected for histopathological evaluation. The resected tumors were fixed in 10% formalin and embedded in paraffin. Tumor sections were deparaffinized and rehydrated in xylene and ethanol, and stained with hematoxylin and eosin (HE) or immunostained with antibodies against Ki-67 (Abcam, Cambridge, UK) in combination with diaminobenzidine (DAB, Dako Japan Inc., Kyoto, Japan). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay using a cell death kit (Roche) was performed according to the manufacturer’s instructions. TUNEL-stained area was measured and Ki-67-positive cells were counted using the Image J software. Kidney, intestine, and blood serum samples were collected from mice and evaluated pathologically and biochemically by an outsourced inspection facility (Safety Research Institute for Chemical Compounds Co., Ltd., Sapporo, Japan) for evaluation of the side effects.

Statistical analysis.

Easy R (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a modified version of R commander (The R Foundation for Statistical Computing, Vienna, Austria), which includes statistical functions for biostatistics, was used for all statistical analyses ⁴. Data were compared using Student’s t-test or one-way analysis of variance (ANOVA) with Tukey’s post-hoc test. All data are expressed as mean ± standard error of the mean (SEM). All p-values were two-sided.

Ethics approval

All research was performed in accordance with relevant guidelines and regulations. The study protocol was approved by Kanazawa University Advanced Science Research Centre (Kanazawa, Japan). All procedures for animal studies were performed in accordance with the Japanese national guidelines, and the protocol was approved by the Institute for Experimental Animals, Kanazawa University Advanced Science Research Center. The authors complied with the ARRIVE guidelines.

Induction of PPARγ by zaltoprofen.

Zaltoprofen treatment upregulated the mRNA and protein levels of PPARγ in H-EMC-SS cells as evidenced by qRT-PCR and western blot analysis, respectively, demonstrating that zaltoprofen can induce PPARγ in EMC (Fig. 1a and b). Next, we investigated the induction of Krox20 and C/EBPs, which regulate PPARγ in the normal fat tissue, in zaltoprofen-treated H-EMC-SS cells. Krox20 was significantly upregulated from 3 to 24 h after the initiation of treatment, followed by a significant upregulation of C/EBPβ starting 12 h and of C/EBPα at 24 h, demonstrating the signal pathway of PPARγ induction by zaltoprofen in EMC (Fig. 1c).

Inhibition of cell proliferation by zaltoprofen.

The inhibitory effect of zaltoprofen on the proliferation of H-EMC-SS cells in vitro was examined by the WST-8 and EdU assays. After treatment of H-EMC-SS cells with zaltoprofen (0–800 µmol/L) for 96 h, their growth was significantly reduced in a dose- and time-dependent manner (Fig. 2a). To investigate the PPARγ-dependency, the cell growth was analyzed after downregulation of PPARγ using an shRNA. The growth of H-EMC-SS^shPPARγ cells was significantly increased compared to that of H-EMC-SS^shNT cells with and without 400 µmol/L zaltoprofen treatment (p < 0.001, Fig. 2b). However, zaltoprofen treatment also significantly inhibited the growth of H-EMC-SS^shPPARγ cells (p < 0.001), suggesting that zaltoprofen also exerts PPARγ-independent effects on the growth of cells.

The EdU-positive cell ratio (EdU-incorporated cells/Hoechst 33342-positive nuclei) was calculated, and the PPARγ−dependency of cell proliferation was confirmed by silencing of PPARγ in the cells (Fig. 3). Zaltoprofen significantly and dose-dependently decreased the EdU-positive cell ratio in H-EMC-SS^shNT cells. The EdU-positive cell ratio in H-EMC-SS^shPPARγ cells was significantly higher than that in H-EMC-SS^shNT cells. However, zaltoprofen treatment significantly reduced the EdU-positive ratio of H-EMC-SS^shPPARγ cells in a dose-dependent manner. These data suggest that proliferation of EMC cells is mediated by PPARγ and the antiproliferation effect of zaltoprofen on EMC cells is both PPARγ-dependent and -independent.

Expression of cell cycle checkpoint proteins, namely p21, p27, and p53, which regulate abnormal cycling of cancer cells, was measured by western blot analysis in both H-EMC-SS^shNT and H-EMC-SS^shPPARγ cells (Fig. 4). Zaltoprofen treatment induced the expression of p21, p27, and p53 in H-EMC-SS^shNT cells but not in H-EMC-SS^shPPARγ cells, demonstrating that zaltoprofen could induce cell-cycle checkpoint proteins in a PPARγ-dependent manner in EMC cells.

Inhibitory effects of zaltoprofen on xenograft tumors in mice.

The effect of zaltoprofen on xenograft tumors and its safety were tested using xenograft mouse models subcutaneously implanted with H-EMC-SS cells. We checked the effect of 400 µmol/L of zaltoprofen, which is an effective concentration in vitro, and equivalent to 30 mg/kg in vivo. Zaltoprofen (30 mg/kg) treatment significantly inhibited the tumor growth at all time points compared with that in the vehicle group (p = 0.032, Fig. 5a). CT images of the zaltoprofen-treated tumor at the treatment end point showed decreased tumor size and increased calcification inside the tumor (Fig. 5b and c). Histopathological analyses were conducted on excised tumors. HE-stained sections showed that tumors in the vehicle group consisted of viable highly dense tumor cells, whereas zaltoprofen-treated tumors had scattered scar areas with infiltration of inflammatory cells (Fig. 5d and e). TUNEL-positive tumors in zaltoprofen-treated group were significantly larger than those in the vehicle group (p = 0.024, Fig. 6a-c), demonstrating that zaltoprofen induced necrosis of tumors in the H-EMC-SS xenograft models. In contrast, tumors in the zaltoprofen-treated group had significantly lower Ki-67 positive cells compared with those in the vehicle group (p < 0.001, Fig. 6d-f), demonstrating that zaltoprofen inhibited the cell cycle in the xenograft tumors. The body weights in both vehicle- and zaltoprofen-treated groups post-administration were higher than the pre-administration weights (p = 0.005 and p = 0.014, respectively; Fig. 7. Generally, long-term administration of NSAIDs can cause kidney injury, although there was no abnormal renal function, as evident from the absence of abnormalities in the levels of blood urea nitrogen and serum creatinine, or pathological abnormalities in renal autopsy in both the groups. In addition, no hepatotoxicity was detected in the analysis of serum total bilirubin in either of the groups. There were no other observable side effects or deaths in either of the mouse groups.

We found that zaltoprofen induced the expression of PPARγ in human EMC cells, H-EMC-SS, in a dose-dependent manner by inducing KROX20, C/EBPβ, and C/EBPα in that order (Fig. 1). Zaltoprofen treatment induced the expression of cell cycle checkpoint proteins, p21, p27, and p53, and significantly inhibited the proliferation of H-EMC-SS cells (Fig. 2–4). The knockdown of PPARγ did not completely cancel the antitumor effect of zaltoprofen (Fig. 2b and Fig. 3), suggesting that zaltoprofen affects cell proliferation in both PPARγ-dependent and -independent manner. The antiproliferative effects of zaltoprofen on H-EMC-SS cells were demonstrated in vivo (Fig. 5–7).

The role of PPARγ in malignant tumor biology has been studied, and PPARγ ligands are known to elicit antitumor effects in cancer cells ^3,5−7. Several drugs, including the antidiabetic and antihypertensive ones, and NSAIDs, are potential PPARγ ligands. Among NSAIDs, indometacin, oxaprozin, diclofenac, and zaltoprofen are reported to be potential PPARγ ligands ^8–10. In the present study, we demonstrate, for the first time, that zaltoprofen induces PPARγ, and elicits antitumor effects against musculoskeletal tumors, both in vitro and in vivo.

C/EBPs form a family of six homologous transcription factors that are involved in regulating cellular proliferation and differentiation, metabolism, and immunity in various organs ^11,12. C/EBPβ and C/EBPδ are induced early during adipogenesis and are involved in the induction of PPARγ ^13,14. C/EBPα is induced at a later stage, which is almost the same stage at which PPAR is induced, and is activated in mature adipocytes ^15,16. C/EBPα promotes adipogenesis both with and without PPARγ induction ^15,16. Krox20, also referred to as early growth response protein 2 (EGR2), a transcription regulatory factor, is an upstream regulator of C/EBPs and one of the earliest factors to be induced during adipogenesis ^17,18. Herein, we demonstrate that, in H-EMC-SS cells, zaltoprofen induces Krox20 at the earliest stage, followed by C/EBPβ and C/EBPα, but not C/EBPδ. This pathway could be one of the mechanisms for the induction of PPARγ by zaltoprofen in H-EMC-SS cells, but it needs to be confirmed for the other malignant cells.

The cyclin-dependent kinase (CDK) inhibitors, p21 and p27, are essential tumor suppressor proteins that play significant roles in the inhibition of the growth of cancer cells ¹⁹. It has been reported that activation of PPARγ induces cell cycle arrest through the upregulation of p21 and p27 in various cancer cells, such as hepatic cell carcinoma ^20–22, melanoma ²³, and pancreatic carcinoma ²⁴. The tumor suppressor protein, p53, also regulates the cell cycle by repairing DNA, inducing apoptosis, and inducing p21 and p27; thus, p21, p27, and p53 interact in complex manner to suppress the cell cycle ²⁵. In the present study, p21, p27, and p53 were induced in H-EMC-SS cells upon zaltoprofen treatment in a PPARγ-dependent manner and inhibited the proliferation of cells. Whether zaltoprofen induces p21 or p27 in a p53-dependent or -independent manner should be examined in future studies. In addition, the PPARγ-independent antitumor effects of zaltoprofen, as evident in the WST-8 and EdU assays in the present study, would require further confirmation in future studies.

In the present study, we have demonstrated the in vivo antiproliferative effect of zaltoprofen on musculoskeletal tumor cells for the first time. The downregulation of Ki-67, a proliferation marker expressed during the active cell-cycle phases, in zaltoprofen-treated tumors indicates the inhibition of cell cycle in vivo by zaltoprofen, which corroborates with the in vitro results. There were more TUNEL-stained cells in zaltoprofen-treated tumors, indicating that zaltoprofen could induce necrosis of tumor cells. The good tolerability of zaltoprofen, which was revealed by the absence of major complications, including body weight loss, nephrotoxicity, and hepatotoxicity, should be useful in carrying out further analysis on the efficacy of zaltoprofen against musculoskeletal tumors for potential clinical application.

In conclusion, we demonstrate that zaltoprofen induces Krox20, C/EBPβ, C/EBPα, and PPARγ, and subsequently p21, p27, and p53 in EMC cells, and contributes to the inhibition of cell proliferation. The antiproliferative effect of zaltoprofen is also demonstrated in vivo with good tolerability. These results suggest that zaltoprofen could be a novel and effective treatment for EMC.

Acknowledgements

This work was supported by JSPS KAKENHI Grant Number JP17K16682.

Author contributions

T.H. designed research, conducted experiments, analyzed and interpreted data, and wrote the manuscript. A.T. conceived and designed research, and approved the manuscript. S.M. and A.H. supported experiments, interpreted data, and approved the manuscript. N.Y. and K.H. supported experiments and approved the manuscript. Y.Y. interpreted data, and reviewed and approved the manuscript. H.T. conceived research and approved the manuscript.

Competing interests

The authors declare no competing interests.

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No competing interests reported.

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A Nonsteroidal Anti-inflammatory Drug, Zaltoprofen, Inhibits the Growth of Extraskeletal Chondrosarcoma Cells by Inducing PPARγ, p21, p27, and p53

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Reagents.

Cell culture.

RNA interference.

RT-qPCR.

Western blot analysis.

Water-soluble tetrazolium salt-8 (WST-8) assay.

5-ethynyl-2′-deoxyuridine (EdU) proliferation assay.

Animal experiments.

Statistical analysis.

Ethics approval

Results

Induction of PPARγ by zaltoprofen.

Inhibition of cell proliferation by zaltoprofen.

Inhibitory effects of zaltoprofen on xenograft tumors in mice.

Discussion

Declarations

Acknowledgements

Author contributions

Competing interests

References

Additional Declarations

Status:

Version 1