Ma XM, Yu H, Huai N. Peroxisome proliferator-activated receptor-γ is essential in the pathogenesis of gastric carcinoma. World J Gastroenterol 2009; 15(31): 3874-3883 [PMID: 19701967 DOI: 10.3748/wjg.15.3874]
Corresponding Author of This Article
Xiu-Mei Ma, Professor, MD, Department of Pathology, The First Affiliated Hospital of Inner Mongolia Medical College, Huhhot 010059, Inner Mongolia Autonomous Region, China. maxiumei0471@yahoo.com.cn
Article-Type of This Article
Original Articles
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Xiu-Mei Ma, Department of Pathology, The First Affiliated Hospital of Inner Mongolia Medical College, Huhhot 010059, Inner Mongolia Autonomous Region, China
Xiu-Mei Ma, Department of Pathology, School of Basic Medical Sciences of Inner Mongolia Medical College, Huhhot 010059, Inner Mongolia Autonomous Region, China
Hong Yu, Na Huai, The Graduate Faculty of Inner Mongolia Medical College, Huhhot 010059, Inner Mongolia Autonomous Region, China
ORCID number: $[AuthorORCIDs]
Author contributions: Ma XM performed the experiments and wrote the manuscript; Yu H and Huai N performed the statistical analyses.
Correspondence to: Xiu-Mei Ma, Professor, MD, Department of Pathology, The First Affiliated Hospital of Inner Mongolia Medical College, Huhhot 010059, Inner Mongolia Autonomous Region, China. maxiumei0471@yahoo.com.cn
Telephone: +86-471-6637678
Received: March 14, 2009 Revised: July 7, 2009 Accepted: July 14, 2009 Published online: August 21, 2009
Abstract
AIM: To investigate whether peroxisome proliferator-activated receptor γ (PPAR-γ) is expressed in human gastric carcinoma and whether PPAR-γ is a potential target for gastric carcinoma therapy.
METHODS: PPAR-γ protein in gastric carcinoma was examined by immunohistochemistry. In the gastric carcinoma cell line MGC803, PPAR-γ, survivin, Skp2 and p27 protein and mRNA were examined by Western blotting and real-time reverse transcription-polymerase chain reaction, respectively; proliferation was examined by MTT; apoptosis was examined by chromatin staining with Hoechst 33342 and fluorescence activated cell sorting (FACS). and cell cycle was examined by FACS; the knockdown of PPAR-γ was done by RNA interference.
RESULTS: A high level of expression of PPAR-γ was observed in human gastric carcinoma and in a human gastric carcinoma cell line MGC803. The PPAR-γ agonist 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) inhibited growth, and induced apoptosis and G1/G0 cell cycle arrest in MGC803 cells in a concentration-dependent and time-dependent manner. The effect of 15d-PGJ2 on MGC803 cells was not reversed by the selective and irreversible antagonist GW9662 for PPAR-γ. Furthermore, survivin and Skp2 expression were decreased, whereas p27 expression was enhanced following 15d-PGJ2 treatment in a dose-dependent manner in MGC803 cells. Interestingly, we also found that small interfering RNA for PPAR-γ inhibited growth and induced apoptosis in MGC803 cells. The inhibition of PPAR-γ function may be a potentially important and novel modality for treatment and prevention of gastric carcinoma.
CONCLUSION: A PPAR-γ agonist inhibited growth of human gastric carcinoma MGC803 cells by inducing apoptosis and G1/G0 cell cycle arrest with the involvement of survivin, Skp2 and p27 and not via PPAR-γ.
Citation: Ma XM, Yu H, Huai N. Peroxisome proliferator-activated receptor-γ is essential in the pathogenesis of gastric carcinoma. World J Gastroenterol 2009; 15(31): 3874-3883
Gastric carcinoma is the second most common cancer in the world and kills more than 600 000 people annually[1]. At present, the management of gastric carcinoma mainly includes surgery and chemotherapy, but the curative effect of the existing chemotherapeutic drugs is not effective and they have numerous side effects. Many studies have been performed to search for therapeutic targets and drugs capable of preventing and treating gastric carcinoma and other malignancies.
Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors, initially described as molecular targets for compounds, which induce peroxisomal proliferation[2]. Up to now, 3 different isotypes of PPARs have been identified in various species, being the products of distinct genes and generally designated as PPAR-α, PPAR-β/δ, and PPAR-γ[23]. PPAR-γ has been extensively studied. Similarly to other members of the nuclear receptor gene family, PPAR-γ is an agonist-activated transcription factor[34]. PPAR-γ heterodimerizes with retinoid X receptor to bind to the PPAR response element, leading to the transcription of downstream genes[5]. Several specific agonists have been found, such as the thiazolidinediones (including pioglitazone, rosiglitazone, and troglitazone), 15-deoxy-Δ12,14-prostaglandin-J2 (15d-PGJ2), and certain polyunsaturated fatty acids. GW9662 has been shown to be a selective and irreversible antagonist of PPAR-γ, and irreversibly binds within the agonist binding domain through covalent modification of a cysteine residue[6].
PPAR-γ is predominately expressed in adipose tissue and plays a central role in adipocyte differentiation and insulin sensitivity[7]. Recent studies have shown that, in addition to its classic role, PPAR-γ is implicated as a putative therapeutic target for cancer in a variety of tumors as several observations suggest that stimulation of PPAR-γ function may inhibit carcinogenesis and tumor cell growth[89]. However, the exact role of PPAR-γ on carcinogenesis and tumor cell growth is still unclear[10–14]. Recent investigations by Morita et al[15] and Konturek et al[16] have shown that PPAR-γ is implicated in Helicobacter pylori-related gastric carcinogenesis, and that PPAR-γ agonists may have potential in a cancer therapeutic role.
We here demonstrate that PPAR-γ may be involved in gastric carcinogenesis, and that the inhibition of PPAR-γ may be of benefit in the treatment of gastric carcinoma.
MATERIALS AND METHODS
Chemicals
PPAR-γ agonist, 15d-PGJ2 and antagonist GW9662 were purchased from Cayman chemical (Ann Arbor, M1). Other drugs were reagent grade.
Tissue samples
One hundred and thirty eight samples of surgically-resected primary gastric carcinoma tissues, 138 samples of paired adjacent mucosa (2-5 cm from the margin of the gastric carcinoma) and 138 samples of paired normal mucosa (histologically proven) at the surgical margin (at least 5 cm from the margin of the gastric carcinoma) tissues were obtained from the First Affiliated Hospital of Inner Mongolia Medical College in China from October 2004 to January 2005. All gastric carcinoma patients underwent total or subtotal gastrectomy, and no patient received any treatment for cancer before surgery. The patient series included 111 males and 27 females with a mean age of 58.5 years and a median age of 60 years (range, 36-78). After surgery, the gastric specimens were fixed in 10% neutral buffered formalin and embedded in paraffin for immunohistochemistry. The clinical stage of all gastric carcinoma specimens were determined according to new TNM criteria published by the Union International Centre Cancer in 1997. All pathological diagnoses were performed by 2 independent pathologists.
Immunohistochemical staining of PPAR-γ
Sections of paraffin-embedded tissues (4 μm) were mounted on glass slides. The intracellular expression of PPAR-γ was detected using a PPAR-γ specific mAb (sc-7372, E-8, Santa Cruz Biotechnology, CA) with standard non-biotin horseradish peroxidase (HRP) 2-step immunostaining (Zymed) according to the manufacturer’s instructions. The immunoreactive products were visualized using 3,3’-diaminobenzidine (DAB)/H2O2.
Cell culture and treatment with PPAR-γ agonists and antagonists
We used the human gastric carcinoma cell line MGC803 from Beijing Tumor Institute. It was maintained in RPMI1640 containing 10% fetal bovine serum (FBS) at 37°C under 5% CO2. A PPAR-γ agonist or antagonist were added at the time of replating.
Cell survival assay
Gastric carcinoma cells were treated with a PPAR-γ agonist or antagonist dissolved in DMSO for 8, 18 and 24 h in culture medium in 96-well plates. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma] solution was then added to the well with gentle pipetting. After 4 h, the supernatant was removed after centrifugation at 1000 r/min for 5 min at room temperature, and 180 μL DMSO was added to each well to solubilize the crystal products by shaking for 10 min. Absorbance at 570 nm was measured with an ELISA reader. The negative control had medium without serum and cells, and was used as the zero point of absorbance. Cell vitality of the control group was 100%. The inhibitory rate (IR) of cells was calculated according to the equation as follows: IR (%) = [1-(A570 nm absorbance in 15d-PGJ2 treated group/A570 nm absorbance in control group)] × 100%. Each assay was performed in triplicate.
Apoptosis analysis
Apoptosis was analyzed by 2 different methods. First, chromatin staining with Hoechst 33342 was carried out. Briefly, MGC803 cells treated with 15d-PGJ2 were cultured on Laboratory-Tek Chamber Slides for 24 h. The cells were then fixed with methanol for 10 min and rinsed. Chromatin staining was done with Hoechst 33342 (Sigma) to detect nuclear condensation. Secondly, cell apoptosis was measured by fluorescence activated cell sorting (FACS) using the Annexin-FITC Apoptosis Detection Kit according to the manufacturer’s protocol. The apoptotic rate was calculated as the percentage of annexin V-positive and propidium iodide (PI)-negative cells divided by the total number of cells in the gated region. The data was pooled from 3 independent experiments.
Cell cycle examination
The effect of 15d-PGJ2 on the cell cycle was examined with PI staining by FACS using Epics-XL (Beckmam Coulter).
Western blotting analysis
Cell total protein was extracted from MGC803 cells using CytoBuster™ Protein Extraction Reagent (Novagen). The concentration of the protein was determined with Coomassie brilliant blue G-250. Cell total protein (50 μg) was separated by 12% SDS-PAGE. After electrophoresis, the proteins were transferred to a polyvinylidene fluoride membrane, blocked overnight in Tris buffered saline with 10% skim milk at 4°C. PPAR-γ (Santa Cruz), Skp2 (Zymed), p27 (Santa Cruz) and β-actin (Santa Cruz) were detected using primary mouse mAb, and survivin (Santa Cruz) was detected using primary rabbit polyclonal Ab at a dilution of 1:2000, followed by HRP-conjugated anti-mouse IgG or anti-rabbit IgG at a dilution of 1:1500. Immunocomplexes were visualized using DAB. β-actin was used as the internal control.
Total RNA was extracted from MGC803 cells using TRIzol reagent (Gibco, USA). The resultant RNA samples were quantified using a spectrophotometer at a wavelength of 260 nm. The integrity of the isolated RNA samples was analyzed electrophoretically on agarose gel, followed by staining in ethidium bromide. An aliquot of 2 μg of total RNA from each sample was reversely transcribed and amplified using RT-PCR Enzyme Mix Kit (Sino-American Biotechology Co.). For detection of human PPAR-γ mRNA, a combination of a sense primer (5'-TCTCTCCGTAATGGAAGACC-3') and an antisense primer (5'-GCATTATGAGACATCCCCCAC-3') were used. The reverse transcription was performed for 1 h at 37°C and amplification was done by initial denaturation at 95°C for 2 min, 40 cycles of amplification (95°C for 40 s, 55°C for 50 s, and 72°C for 50 s) and a final extension step of 7 min at 72°C was carried out in a 30 μL mixture containing 2 μg of total RNA, 25 pmol/L each of the sense and antisense primers, 2.0 mmol/L MgCl2, 0.1 mmol/L dNTP, 1.5 μL 20 × buffer without Mg2+, 2 μL RT-PCR Enzyme Mix and double-distilled H2O. β-actin sense primer, 5'-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3', and antisense primer 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3' were used. The reverse transcription was done for 1 h at 37°C and amplification was performed by initial denaturation at 94°C for 5 min, 35 cycles of amplification (95°C for 15 s, 60°C for 1 min, and 72°C for 30 s) and a final extension step of 7 min at 72°C was carried out in a 30 μL mixture containing the same components as above. The length of the β-actin PCR product was 838 bp and was the internal control. The PCR reaction products were separated electrophoretically in a 2% agarose gel and stained with ethidium bromide for 40 min at 80 V, and observed and photographed. All primers were synthesized by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd.
Construction of small interfering RNA (siRNA) expression vectors
To further elucidate the effect of PPAR-γ in gastric carcinoma, we applied the siRNA targeting approach. We used shRNA™ vector (provided by Wuhan Genesil Biotechnology Co., Ltd.) in which shRNA expression is driven by the U6 RNA promoter to produce small and haired RNA transcripts. We generated 3 different shRNA™ vectors and an empty vector used as a control. The oligonucleotides 5'-GATCCGAACAGATCCAGTGGTTGCTTCAAGA-CGGCAACCACTGGATCTG-TTCTTTTTTGTCGACA-3', 3'-GCTTGTCTAGGTCACCAACGAAGTTCTGCCGTTGGTGACCTAGACAAGAAAAAACAGCTGTTCGA-5' were used to generate anti-heparanase shRNA™ pSi1, and oligonucleotides 5'-GATCCGTCAAA-GTGGAGCCTGCATCTTCAAGACGGATGCAGGCTCCACTTTGATTTTTTGTCGACA-3', 3'-GCAGTTTCACCTCGGACGTAGAAGTTCTGCCTACGTCCGAGGTGAAACTAAAAAACAGCTGTTCGA-5' were used to generate anti-heparanase shRNA™ pSi2. Oligonucleotides 5'-GATCCG-ACAACCTGCTACAAGCCCTTCAAGACGGG-GCTTGTAGCAGGTTGTCTTTTTTGTCGACA-3', 3'-GCTGTTGGACGATGTTCGGGAAGTTCTGCCCCGAACATCGTCCAACAGAAAAAACAGCTGTTCGA-5' were used to generate anti-heparanase shRNA™ pSi3.
Transfection
For the transfection, pSi1, pSi2 and pSi3 (1.0 μg) were added to RPMI1640 medium (without FBS) containing cation liposome vector (2.0 μL) and allowed to incubate for 20 min at room temperature to produce the transfection mixture. The transfection mixture was then added to the cells (1 × 105 per dish) in the serum-free medium. Six hours after the start of transfection, the medium was changed to RPMI1640 medium containing 10% FBS. RNA interference effect was examined by RT-PCR, Western blotting and MTT assay. Cell apoptosis determination was carried out at the final step.
Statistical analysis
The results were expressed as mean ± SD. Statistical analysis was performed using the Student’s t-test, χ2 test or one-way ANOVA and subsequent Fisher’s LSD test. P < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS11.0 software.
RESULTS
Enhanced PPAR-γ expression in gastric carcinoma tissue and MGC803 cells
The positive rate of PPAR-γ protein expression was significantly higher in gastric carcinoma (75.0%) than in paired mucosa adjacent to gastric carcinoma (27.5%) and in normal gastric mucosa at the surgical margin of gastric carcinoma (13.0%) (P < 0.001). Mucosa adjacent to gastric carcinoma had significantly higher expression than normal gastric mucosa at the surgical margin of gastric carcinoma (P < 0.05 and P < 0.001) (Figure 1). The rate of PPAR-γ protein expression was positively associated with histologic type and negatively associated with invasive depth, vessel invasion, lymph node metastasis, distant metastasis and clinical stage (P < 0.05) of gastric carcinoma. The rate of PPAR-γ protein expression was significantly higher in intestinal-type gastric carcinoma (95.2%) than in diffuse-type gastric carcinoma (58.7%) (P < 0.001) (Table 1). Both PPAR-γ mRNA and protein showed positivity in the human gastric carcinoma cell line MGC803 (Figure 2).
Table 1 Correlation of of PPARγ protein expression in gastric carcinoma with clinicopathological features.
Figure 1 Peroxisome proliferator-activated receptor-γ (PPAR-γ) protein in the cytoplasm of gastric carcinoma cells by immunohistochemistry (3,3’-diaminobenzidine and hematoxylin staining × 400).
A: Well differentiated gastric carcinoma; B: Poorly differentiated gastric carcinoma.
Figure 2 PPAR-γ mRNA and protein expression in gastric carcinoma cell line MGC803.
A: PPARγ mRNA expression examined in MGC803 cells by reverse transcriptase-polymerase chain reaction. M: 100 bp DNA marker and 500 bp was the brightest; 1: MGC803 cells. B: PPAR-γ protein expression examined by Western blotting in MGC803 cells.
Effect of 15d-PGJ2, GW9662 and 15d-PGJ2 + GW9662 on growth of MGC803 cells
Treatment with a concentration of 0.01, 0.1 and 1 μmol/L 15d-PGJ2 did not affect MGC803 cell growth. The inhibition of proliferation of MGC803 cells by 5, 10, 20, 30, and 40 μmol/L 15d-PGJ2 was significantly higher than that of 0 μmol/L (P < 0.001) (Table 2). Correspondingly, inhibition was also enhanced with increasing 15d-PGJ2 concentrations. Inhibition was 5.98% ± 1.41%, 13.64% ± 0.69%, and 34.64% ± 0.99% after 8, 18, and 24 h, respectively, following 30 μmol/L 15d-PGJ2 addition. It was significantly higher at 18 h than 8 h (P < 0.001), and it was significantly higher at 24 h than 18 h (P < 0.001); it was enhanced over a prolonged time. Thus 15d-PGJ2 inhibited proliferation of MGC803 cells in a concentration-dependent and time-dependent manner (Figure 3A). The proliferation inhibition rate of MGC803 cells by GW9662 treatment for 24 h, showed no difference between 0.01, 0.1 and 1 μmol/L and 0 μmol/L concentrations (all P > 0.05). Inhibition of proliferation was significantly higher at 2.5 and 5 μmol/L compared to 0 μmol/L (all P > 0.05). The proliferation inhibition rate of MGC803 cells by 15d-PGJ2 and GW9662 combination treatment for 24 h showed no difference between the 0.01, 0.1 and 1 μmol/L GW9662 and 5, 10, 20, 30 and 40 μmol/L 15d-PGJ2 combination groups and the 5, 10, 20, 30 and 40 μmol/L 15d-PGJ2 group (Table 3).
Table 2 Effect of various concentrations of 15-deoxy-Δ12,14-prostaglandin-J2 (15d-PGJ2) on proliferation, apoptosis and cell cycle of gastric carcinoma MGC803 cells after 24 h [(mean ± SD) %].
Table 3 Effect of GW9662 in various concentrations on the proliferation of MGC803 cells and on 15d-PGJ2 inhibition of proliferation of MGC803 cells after 24 h [(mean ± SD) %].
Figure 3 15d-PGJ2 inhibited proliferation (A) and induced apoptosis (B) of MGC803 cells in a concentrationdependent and time-dependent manner.
Effect of 15d-PGJ2 on apoptosis of MGC803 cells
15d-PGJ2 is capable of inducing MGC803 cell apoptosis as seen by morphological observation (Figure 4) and flow cytometry (Figure 5). The apoptosis rate of MGC803 cells of 5, 10, 20, 30, and 40 μmol/L 15d-PGJ2 was significantly higher than that of 0 μmol/L (P < 0.001) (Table 1); it was enhanced with increasing concentrations of 15d-PGJ2. The apoptosis rate was 6.58% ± 0.62%, 15.99% ± 1.73%, and 38.23% ± 1.36% at 8, 18 and 24 h, respectively, following 30 μmol/L 15d-PGJ2 addition, and increased with time. Thus, 15d-PGJ2 induced MGC803 cell apoptosis in a concentration-dependent and time-dependent manner (Figure 3B).
Figure 4 Morphological changes of MGC803 cells 24 h after treatment with 30 μmol/L 15d-PGJ2.
Cell shapes were observed by fluorescence microscopy (Hoechst 33342 staining, × 400). Nuclear chromatin condensation, marginalization of nuclear chromatin and half moon formation, nuclear fragments with bright chromatin and apoptotic bodies were easily identified in some cells.
Figure 5 Apoptosis rate of MGC803 cells detected by flow cytometry 24 h after treatment with 15d-PGJ2 at various concentrations.
A: 0 μmol/L; B: 5 μmol/L; C: 10 μmol/L; D: 20 μmol/L; E: 30 μmol/L; F: 40 μmol/L.
Effect of 15d-PGJ2 on cell cycle of MGC803 cells
15d-PGJ2 at 30 μmol/L significantly increased the proportion of MGC803 cells in the G0/G1 phase (P < 0.001) and significantly decreased the proportion in the S and G2/M phases (P < 0.001 and P < 0.01, respectively) (Figure 6).
Figure 6 Cell cycle distribution of MGC803 cells by flow cytometry 24 h after treatment with 15d-PGJ2 at various concentrations.
15d-PGJ2 at 30 μmol/L could significantly increase the proportion of cells in the G0/G1 phase (P < 0.001) and decrease the proportion in the S and G2/M phases in MGC803 cells (P < 0.001 and P < 0.01).
Effect of 15d-PGJ2 on survivin, Skp2 and p27 expression in MGC803 cells
Survivin and Skp2 protein were decreased and p27 protein was increased as the concentration of 15d-PGJ2 increased as shown by Western blotting (Figure 7).
Figure 7 Survivin, Skp2 and p27 protein expression by Western blotting in MGC803 cells 24 h after treatment with 15d-PGJ2 at various concentrations.
Lane 1: 0 μmol/L; Lane 2: 5 μmol/L; Lane 3: 20 μmol/L; Lane 4: 30 μmol/L. Survivin and Skp2 protein decreased and p27 protein increased with increasing concentrations of 15d-PGJ2.
Effect of knockdown of PPAR-γ by RNAi
To examine whether the inhibitory effect of PPAR-γ agonist 15d-PGJ2 on the growth of MGC803 cells was via a PPAR-γ specific pathway and influenced PPAR-γ expression in the tumor cells, we used the RNAi approach for PPAR-γ. Efficiency of transfection approximate 80%) was evaluated by fluorescence microscopy 48 h after transfection of a vector containing the gene encoding green fluorescent protein (Figure 8). MGC803 cells were transiently transfected with pSi1, pSi2 and pSi3 or empty vectors by cation liposome vector, and the cells were tested for PPAR-γ expression 48 h later. Of the cells transfected with pSi1, pSi2 and pSi3, pSi1 was most effective in silencing PPAR-γ mRNA and protein expression in MGC803 cells, as determined by semiquantitative RT-PCR (Figure 9A) and Western blotting (Figure 9B). PPAR-γ specific RNAi effectively decreased PPAR-γ mRNA and protein level in the MGC803 cells and showed inhibition of cell growth (Figure 10A) and induction of cell apoptosis (Figure 10B).
Figure 9 PPARγ after pSi transfection.
A: PPARγ mRNA expression in various cells detected by RT-PCR 48 h after transfection; B: PPARγ protein expression in various cells 48 h after transfection as detected by Western blotting. Lane M: 100 bp DNA marker, 500 bp was the brightest; Lane 1: MGC803-N (normal MGC803 cells); Lane 2: MGC803-HK (MGC803 cells transfected by empty vector); Lane 3: MGC803-pSi3 (MGC803 cells transfected by pSi3 vector); Lane 4: MGC803-pSi2 (MGC803 cells transfected by pSi2 vector); Lane 5: MGC803-pSi1 (MGC803 cells transfected by pSi1 vector). The effect of pSi1 was best in silencing PPAR-γ mRNA and protein expression in MGC803 cells.
Figure 10 Proliferative inhibition and apoptosis rate of MGC803-Y1 and of MGC803-HK cells after transfection.
The proliferative inhibition (A) and apoptosis (B) rate of MGC803-Y1 cells was higher than that of MGC803-HK cells at 12-72 h after transfection.
DISCUSSION
Recently, the potential of PPAR-γ as a target for the prevention and treatment of cancer has been widely studied[17]. However, the potential therapeutic role of PPAR-γ agonists has been questioned, based on contradictory results. In experiments using animal models of colon cancer, PPAR-γ agonists increased the development of colon tumors[10]. This contradictory result was supplemented by a recent report using transgenic mice that expressed a constitutive active form of PPAR-γ in mammary glands, and showed that PPAR-γ signaling accelerated tumor development in mammary glands[18]. The actual role of PPAR-γ in cancer has been complicated by recent findings that PPAR-γ agonists affect cancer cells independent of PPAR-γ[19–22], and silencing of PPAR-γ and PPAR-γ antagonists have been shown to inhibit cancer cell growth[1314]. To date, the role of PPAR-γ in gastric carcinogenesis remains unclear.
In the present study, PPAR-γ was found to be expressed at higher levels in gastric carcinoma than in paired mucosa adjacent to gastric carcinoma, and in both tissues the PPAR-γ levels were higher than in their paired normal gastric mucosa at the surgical margin of the gastric carcinoma. Similar effects were also observed in gastric cancer in another study[16]. In addition, we used the siRNA approach to confirm the effect of PPAR-γ on gastric carcinoma. The result showed that silencing PPAR-γ clearly inhibited the growth and induced apoptosis of MGC803 cells. Similar effects were also observed in other cancers such as hepatocellular carcinoma[13]. These results indicated that PPAR-γ may be involved in gastric carcinogenesis.
Here, we clearly showed that the PPAR-γ agonist 15d-PGJ2 inhibited growth of cultured gastric cancer MGC803 cells, and our study also demonstrated that the PPAR-γ antagonist GW9662 did not interfere with this effect and that 2.5 μmol/L GW9662 inhibited growth of MGC803 cells. Furthermore, PPAR-γ siRNA markedly inhibited the growth of MGC803 cells in our study. These results indicated that 15d-PGJ2 inhibited growth of cultured gastric carcinoma MGC803 cells by a PPAR-γ-independent pathway. These results also suggested that PPAR agonists may be useful in the chemoprevention or chemotherapy of gastric malignancies. Additionally, some recent studies reported that the biological effect of PPAR-γ agonists is independent of PPAR-γ[1923–28], but other research concluded that 15d-PGJ2 inhibited growth of cancer cells and gastric cancer cells by activating the PPAR-γ pathway[29–32]. What is the reason for the discrepancies between our data and the data found in other reports? This may be attributed to the fact that some investigators did not study the effect of PPAR antagonists on tumor cell growth and on the effect of PPAR agonists, as they only used PPAR agonists.
The inhibition of growth of cultured gastric cancer MGC803 cells by PPAR-γ agonists is mainly a result of apoptosis, as evidenced by the data for nuclear condensation by chromatin staining with Hoechst 33342 and annexin V-FITC staining through FACS analysis. Mechanisms may exist by which apoptosis is induced by PPAR agonists. Furthermore, the inhibition of cell growth and induction of apoptosis by PPAR-γ agonists were time- and concentration-dependent.
Survivin is an inhibitor of apoptosis, and is expressed during embryonal development but lacks expression in terminally differentiated adult tissues. Interestingly, it becomes re-expressed in transformed cell lines and in a variety of human tumors, and deserves growing attention as “an ideal target” for cancer therapy. Survivin is involved in the control of apoptosis and directly inhibits caspase 3 and 7 activities. Survivin is mostly expressed in cell cycle G2/M and lacks expression in G1/G0[33]. During tumorigenesis, survivin expression is negatively correlated with apoptosis but positively correlated with proliferation and angiogenesis[34]. Our study showed that the levels of survivin protein were decreased with the increase in 15d-PGJ2 concentration, and were inversely associated with the apoptosis induced by 15d-PGJ2. These data suggest that 15d-PGJ2 may inhibit survivin expression to induce apoptosis in gastric carcinoma MGC803 cells.
Our study showed, by flow cytometry, that 15d-PGJ2 induced MGC803 cell arrest in the G1/G0 cell cycle phase and reduced the percentage of cells in the S and G2/M cell cycle phases. This may be another reason for inhibition of the growth of gastric carcinoma MGC803 cells by 15d-PGJ2. Additionally, this may be in part the reason that 15d-PGJ2 reduced the level of survivin, although we cannot exclude the possibility that 15d-PGJ2 may have a direct effect on survivin expression. In eukaryotes, progression of the cell cycle is controlled by interactions between cell cycle control proteins (cyclins) and their catalytically active cyclin-dependent kinases (CDKs). The activity of each cyclin-CDK complex is in turn regulated by several different mechanisms, the most important being negative regulation by CDK inhibitors[35]. p27 is an inhibitor of cyclin E-CDK2 and cyclin A-CDK2, which drive cells from the G1 phase to the S phase of the cell division cycle[3637], and p27 is expressed at its highest level in the G1/G0 phase. The amount of p27 is mainly regulated by posttranslational ubiquitin-proteasome-mediated proteolysis[38]. The cell cycle-dependent degradation of p27 is dependent on phosphorylation at Thr187 in the late G1 phase by CDK2, and Thr187 phosphorylation is a necessary prerequisite for the sequential addition of ubiquitin molecules by a ubiquitin ligase complex, SCFskp2, composed of Skp1, Cull, Rbx1, and the F-box protein Skp2[37]. Polyubiquitination of p27 then targets p27 for degradation in the proteasome, thus removing the p27 cell cycle “brake”, allowing cells to transition from G1 to the S phase[39]. Some investigations have shown that Skp2 is a specific substrate-recognition subunit of SCFskp2, the expression of Skp2 is required for the ubiquitination and subsequent degradation of p27 in vitro and in vivo[40–42], and Skp2 knockout cells exhibit p27 at high levels[43]. Skp2 levels are cell cycle regulated, and Skp2 accumulates during the S phase. Inappropriate expression of Skp2 in G0 cells can promote S phase entry concomitant with loss of p27. Recently, the targeted disruption of Skp2 resulted in an accumulation of p27, and cell cycle arrest in the G1 cycle. The level of Skp2 protein was decreased, whereas the level of p27 protein was increased with increasing concentration of 15d-PGJ2 treatment when examined by Western blotting. These data suggested that 15d-PGJ2 induced MGC803 cell arrest in G1/G0 by Down-regulating Skp2 expression and upregulating p27 expression. Furthermore, these results suggested that 15d-PGJ2 may interfere with ubiquitin-proteasome-mediated proteolysis in gastric carcinoma cells.
In conclusion, our study suggests that PPAR-γ may be involved in gastric carcinogenesis, and that 15d-PGJ2 may inhibit the growth of human gastric carcinoma MGC803 cells by inducing apoptosis and G1/G0 arrest, with the involvement of survivin, Skp2 and p27, but via a PPAR-γ-independent pathway.
COMMENTS
Background
Many efforts have been made to search for therapeutic targets and drugs capable of preventing and treating gastric carcinoma and other malignancies. Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors, initially described as molecular targets for compounds, which induce peroxisomal proliferation.
Research frontiers
Three different isotypes of PPARs have been identified in various species, being the products of distinct genes and generally designated as PPAR-α, PPAR-β/δ, and PPAR-γ. PPAR-γ is one that has been extensively studied. As with other members of the nuclear receptor gene family, PPAR-γ is an agonist-activated transcription factor. PPAR-γ heterodimerizes with retinoid X receptor to binding to the PPAR response element, leading to the transcription of downstream genes.
Innovations and breakthroughs
Several specific agonists have been found, such as the thiazolidinediones (including pioglitazone, rosiglitazone, and troglitazone), 15-deoxy-Δ12,14-prostaglandin-J2 (15d-PGJ2), and certain polyunsaturated fatty acids. GW9662 has been shown to be a selective and irreversible antagonist of PPAR-γ, and irreversibly binds within the agonist binding domain through covalent modification of a cysteine residue.
Applications
By understanding how PPAR-γ affects the growth of cells, this study may represent a future strategy for therapeutic intervention in the treatment of patients with gastric carcinoma.
Terminology
PPAR-γ receptors are nuclear hormone receptors, which induce peroxisomal proliferation. 15d-PGJ2 is a specific agonist. Peroxisomal proliferation is thought to be crucial in gastric carcinoma.
Peer review
PPAR-γ receptors are nuclear hormone receptors. The authors examined whether PPAR-γ may affect gastric carcinogenesis, and 15d-PGJ2 may inhibit the growth of MGC803 cells by inducing apoptosis and G1/G0 arrest, but through a PPAR-γ-independent pathway. The result may be useful in treatment of gastric carcinoma.
Footnotes
Peer reviewer: Toru Hiyama, MD, PhD, Health Service Center, Hiroshima University, 1-7-1 Kagamiyama, Higashihiroshima 739-8521, Japan
Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000.Int J Cancer. 2001;94:153-156.
[PubMed] [DOI][Cited in This Article: ]
Willson TM, Brown PJ, Sternbach DD, Henke BR. The PPARs: from orphan receptors to drug discovery.J Med Chem. 2000;43:527-550.
[PubMed] [DOI][Cited in This Article: ]
Feige JN, Gelman L, Michalik L, Desvergne B, Wahli W. From molecular action to physiological outputs: peroxisome proliferator-activated receptors are nuclear receptors at the crossroads of key cellular functions.Prog Lipid Res. 2006;45:120-159.
[PubMed] [DOI][Cited in This Article: ]
Bardot O, Aldridge TC, Latruffe N, Green S. PPAR-RXR heterodimer activates a peroxisome proliferator response element upstream of the bifunctional enzyme gene.Biochem Biophys Res Commun. 1993;192:37-45.
[PubMed] [DOI][Cited in This Article: ]
Leesnitzer LM, Parks DJ, Bledsoe RK, Cobb JE, Collins JL, Consler TG, Davis RG, Hull-Ryde EA, Lenhard JM, Patel L. Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662.Biochemistry. 2002;41:6640-6650.
[PubMed] [DOI][Cited in This Article: ]
Desvergne B, Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism.Endocr Rev. 1999;20:649-688.
[PubMed] [DOI][Cited in This Article: ]
Panigrahy D, Shen LQ, Kieran MW, Kaipainen A. Therapeutic potential of thiazolidinediones as anticancer agents.Expert Opin Investig Drugs. 2003;12:1925-1937.
[PubMed] [DOI][Cited in This Article: ]
Martelli ML, Iuliano R, Le Pera I, Sama' I, Monaco C, Cammarota S, Kroll T, Chiariotti L, Santoro M, Fusco A. Inhibitory effects of peroxisome poliferator-activated receptor gamma on thyroid carcinoma cell growth.J Clin Endocrinol Metab. 2002;87:4728-4735.
[PubMed] [DOI][Cited in This Article: ]
Lefebvre AM, Chen I, Desreumaux P, Najib J, Fruchart JC, Geboes K, Briggs M, Heyman R, Auwerx J. Activation of the peroxisome proliferator-activated receptor gamma promotes the development of colon tumors in C57BL/6J-APCMin/+ mice.Nat Med. 1998;4:1053-1057.
[PubMed] [DOI][Cited in This Article: ]
Saez E, Tontonoz P, Nelson MC, Alvarez JG, Ming UT, Baird SM, Thomazy VA, Evans RM. Activators of the nuclear receptor PPARgamma enhance colon polyp formation.Nat Med. 1998;4:1058-1061.
[PubMed] [DOI][Cited in This Article: ]
Sarraf P, Mueller E, Jones D, King FJ, DeAngelo DJ, Partridge JB, Holden SA, Chen LB, Singer S, Fletcher C. Differentiation and reversal of malignant changes in colon cancer through PPARgamma.Nat Med. 1998;4:1046-1052.
[PubMed] [DOI][Cited in This Article: ]
Masuda T, Wada K, Nakajima A, Okura M, Kudo C, Kadowaki T, Kogo M, Kamisaki Y. Critical role of peroxisome proliferator-activated receptor gamma on anoikis and invasion of squamous cell carcinoma.Clin Cancer Res. 2005;11:4012-4021.
[PubMed] [DOI][Cited in This Article: ]
Morita D, Ichikura T, Mochizuki H. [The expression of PPAR gamma in gastric cancer].Nippon Rinsho. 2001;59 Suppl 4:595-597.
[PubMed] [DOI][Cited in This Article: ]
Konturek PC, Kania J, Kukharsky V, Raithel M, Ocker M, Rembiasz K, Hahn EG, Konturek SJ. Implication of peroxisome proliferator-activated receptor gamma and proinflammatory cytokines in gastric carcinogenesis: link to Helicobacter pylori-infection.J Pharmacol Sci. 2004;96:134-143.
[PubMed] [DOI][Cited in This Article: ]
Sporn MB, Suh N, Mangelsdorf DJ. Prospects for prevention and treatment of cancer with selective PPARgamma modulators (SPARMs).Trends Mol Med. 2001;7:395-400.
[PubMed] [DOI][Cited in This Article: ]
Saez E, Rosenfeld J, Livolsi A, Olson P, Lombardo E, Nelson M, Banayo E, Cardiff RD, Izpisua-Belmonte JC, Evans RM. PPAR gamma signaling exacerbates mammary gland tumor development.Genes Dev. 2004;18:528-540.
[PubMed] [DOI][Cited in This Article: ]
Palakurthi SS, Aktas H, Grubissich LM, Mortensen RM, Halperin JA. Anticancer effects of thiazolidinediones are independent of peroxisome proliferator-activated receptor gamma and mediated by inhibition of translation initiation.Cancer Res. 2001;61:6213-6218.
[PubMed] [DOI][Cited in This Article: ]
Clay CE, Namen AM, Atsumi G, Trimboli AJ, Fonteh AN, High KP, Chilton FH. Magnitude of peroxisome proliferator-activated receptor-gamma activation is associated with important and seemingly opposite biological responses in breast cancer cells.J Investig Med. 2001;49:413-420.
[PubMed] [DOI][Cited in This Article: ]
Clay CE, Monjazeb A, Thorburn J, Chilton FH, High KP. 15-Deoxy-delta12,14-prostaglandin J2-induced apoptosis does not require PPARgamma in breast cancer cells.J Lipid Res. 2002;43:1818-1828.
[PubMed] [DOI][Cited in This Article: ]
Place AE, Suh N, Williams CR, Risingsong R, Honda T, Honda Y, Gribble GW, Leesnitzer LM, Stimmel JB, Willson TM. The novel synthetic triterpenoid, CDDO-imidazolide, inhibits inflammatory response and tumor growth in vivo.Clin Cancer Res. 2003;9:2798-2806.
[PubMed] [DOI][Cited in This Article: ]
Wang M, Wise SC, Leff T, Su TZ. Troglitazone, an antidiabetic agent, inhibits cholesterol biosynthesis through a mechanism independent of peroxisome proliferator-activated receptor-gamma.Diabetes. 1999;48:254-260.
[PubMed] [DOI][Cited in This Article: ]
Castrillo A, Mojena M, Hortelano S, Boscá L. Peroxisome proliferator-activated receptor-gamma-independent inhibition of macrophage activation by the non-thiazolidinedione agonist L-796,449. Comparison with the effects of 15-deoxy-delta(12,14)-prostaglandin J(2).J Biol Chem. 2001;276:34082-34088.
[PubMed] [DOI][Cited in This Article: ]
Okano H, Shiraki K, Inoue H, Yamanaka Y, Kawakita T, Saitou Y, Yamaguchi Y, Enokimura N, Yamamoto N, Sugimoto K. 15-deoxy-delta-12-14-PGJ2 regulates apoptosis induction and nuclear factor-kappaB activation via a peroxisome proliferator-activated receptor-gamma-independent mechanism in hepatocellular carcinoma.Lab Invest. 2003;83:1529-1539.
[PubMed] [DOI][Cited in This Article: ]
Weber SM, Scarim AL, Corbett JA. PPARgamma is not required for the inhibitory actions of PGJ2 on cytokine signaling in pancreatic beta-cells.Am J Physiol Endocrinol Metab. 2004;286:E329-E336.
[PubMed] [DOI][Cited in This Article: ]
Shimada T, Kojima K, Yoshiura K, Hiraishi H, Terano A. Characteristics of the peroxisome proliferator activated receptor gamma (PPARgamma) ligand induced apoptosis in colon cancer cells.Gut. 2002;50:658-664.
[PubMed] [DOI][Cited in This Article: ]
Nikitakis NG, Siavash H, Hebert C, Reynolds MA, Hamburger AW, Sauk JJ. 15-PGJ2, but not thiazolidinediones, inhibits cell growth, induces apoptosis, and causes downregulation of Stat3 in human oral SCCa cells.Br J Cancer. 2002;87:1396-1403.
[PubMed] [DOI][Cited in This Article: ]
Fukuchi K, Date M, Azuma Y, Shinohara M, Takahashi H, Ohura K. Apoptosis in human oral squamous cell carcinomas is induced by 15-deoxy-delta 12,14-prostaglandin J2 but not by troglitazone.J Dent Res. 2003;82:802-806.
[PubMed] [DOI][Cited in This Article: ]
Yoshida K, Hirose Y, Tanaka T, Yamada Y, Kuno T, Kohno H, Katayama M, Qiao Z, Sakata K, Sugie S. Inhibitory effects of troglitazone, a peroxisome proliferator-activated receptor gamma ligand, in rat tongue carcinogenesis initiated with 4-nitroquinoline 1-oxide.Cancer Sci. 2003;94:365-371.
[PubMed] [DOI][Cited in This Article: ]
Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, Altieri DC. Control of apoptosis and mitotic spindle checkpoint by survivin.Nature. 1998;396:580-584.
[PubMed] [DOI][Cited in This Article: ]
O'Connor DS, Grossman D, Plescia J, Li F, Zhang H, Villa A, Tognin S, Marchisio PC, Altieri DC. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin.Proc Natl Acad Sci USA. 2000;97:13103-13107.
[PubMed] [DOI][Cited in This Article: ]
Philipp-Staheli J, Payne SR, Kemp CJ. p27(Kip1): regulation and function of a haploinsufficient tumor suppressor and its misregulation in cancer.Exp Cell Res. 2001;264:148-168.
[PubMed] [DOI][Cited in This Article: ]
Slingerland J, Pagano M. Regulation of the cdk inhibitor p27 and its deregulation in cancer.J Cell Physiol. 2000;183:10-17.
[PubMed] [DOI][Cited in This Article: ]
Pagano M, Tam SW, Theodoras AM, Beer-Romero P, Del Sal G, Chau V, Yew PR, Draetta GF, Rolfe M. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27.Science. 1995;269:682-685.
[PubMed] [DOI][Cited in This Article: ]
Eguchi H, Herschenhous N, Kuzushita N, Moss SF. Helicobacter pylori increases proteasome-mediated degradation of p27(kip1) in gastric epithelial cells.Cancer Res. 2003;63:4739-4746.
[PubMed] [DOI][Cited in This Article: ]
Hershko D, Bornstein G, Ben-Izhak O, Carrano A, Pagano M, Krausz MM, Hershko A. Inverse relation between levels of p27(Kip1) and of its ubiquitin ligase subunit Skp2 in colorectal carcinomas.Cancer. 2001;91:1745-1751.
[PubMed] [DOI][Cited in This Article: ]
Mamillapalli R, Gavrilova N, Mihaylova VT, Tsvetkov LM, Wu H, Zhang H, Sun H. PTEN regulates the ubiquitin-dependent degradation of the CDK inhibitor p27(KIP1) through the ubiquitin E3 ligase SCF(SKP2).Curr Biol. 2001;11:263-267.
[PubMed] [DOI][Cited in This Article: ]
Yang G, Ayala G, De Marzo A, Tian W, Frolov A, Wheeler TM, Thompson TC, Harper JW. Elevated Skp2 protein expression in human prostate cancer: association with loss of the cyclin-dependent kinase inhibitor p27 and PTEN and with reduced recurrence-free survival.Clin Cancer Res. 2002;8:3419-3426.
[PubMed] [DOI][Cited in This Article: ]
Nakayama K, Nagahama H, Minamishima YA, Matsumoto M, Nakamichi I, Kitagawa K, Shirane M, Tsunematsu R, Tsukiyama T, Ishida N. Targeted disruption of Skp2 results in accumulation of cyclin E and p27(Kip1), polyploidy and centrosome overduplication.EMBO J. 2000;19:2069-2081.
[PubMed] [DOI][Cited in This Article: ]
