|Year : 2019 | Volume
| Issue : 1 | Page : 120-125
The administration of peroxisome proliferator-activated receptors α/γ agonist TZD18 inhibits cell growth and induces apoptosis in human gastric cancer cell lines
Yuhong Ma, Bin Wang, Ling Li, Fang Wang, Xichao Xia
Department of Basic Medicine Courses, Nanyang Medical College, Nanyang 473003, PR China
|Date of Web Publication||13-Mar-2019|
Dr. Xichao Xia
Medicine College of Pingdingshan University, Pingdingshan 476000, Henan Province
Source of Support: None, Conflict of Interest: None
Aim of Study: This study is to investigate the effects of a novel peroxisome proliferator-activated receptor (PPAR) α/γ dual agonist TZD18 on cell growth, apoptosis, caspase activity, mitochondrial membrane potential, cytochrome c release, and apoptotic-related protein expression in MKN-45 cells.
Materials and Methods: 3-(4, 5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide assay against various human cancer cell lines was performed to investigate the whether TZD18 could in reduce the proliferation rates of cancer cells. The percentages of apoptotic cells and mitochondrial membrane potential level were determined by flow cytometry. The subcellular localization of cytochrome c was examined by immunofluorescence microscopy. Western blotting assay was performed to reveal the expression of apoptosis-related proteins.
Results: The results showed that the administration of TZD18 could inhibit the growth of MKN-45 cells in a dose- and time-dependent manner. In addition, the apoptotic ratio increased sharply along with a significant increase of caspase activities, mitochondrial membrane potential, and cytochrome c release following TZD18 exposure. The expression of Bax and p27kip1 increased significantly, whereas the expression level of Bcl-2 protein was downregulated.
Conclusion: These results indicated that the administration of PPAR α/γ agonist TZD18 may inhibit cell growth by inducing the apoptotic process in MKN-45 cells.
Keywords: Apoptosis, gastric cancer cell, MKN-45 cells, peroxisome proliferator-activated receptors α/γ dual agonist
|How to cite this article:|
Ma Y, Wang B, Li L, Wang F, Xia X. The administration of peroxisome proliferator-activated receptors α/γ agonist TZD18 inhibits cell growth and induces apoptosis in human gastric cancer cell lines. J Can Res Ther 2019;15:120-5
|How to cite this URL:|
Ma Y, Wang B, Li L, Wang F, Xia X. The administration of peroxisome proliferator-activated receptors α/γ agonist TZD18 inhibits cell growth and induces apoptosis in human gastric cancer cell lines. J Can Res Ther [serial online] 2019 [cited 2019 Dec 6];15:120-5. Available from: http://www.cancerjournal.net/text.asp?2019/15/1/120/208753
| > Introduction|| |
The incidence and mortality of gastric cancer have shown a widespread decrease in recent years; however, gastric cancer is still the second leading cause of cancer death. According to the previous study, gastric cancer is estimated to account for approximately 10% of invasive cancers around the word., Even in Australia where the cancer mortality rates are considered to be relatively low, gastric cancer is one of the leading causes of cancer death raising a wide public concern during the late 20th century.,
In general, chemoprevention plays a potential role in host defense against human cancers through the administration of pharmacologic reagents selectively interacting with the specific molecular targeting ligands such as estrogen, androgen, and retinoid receptor and thereby inhibiting the tumor development processes., For example, the administration of prostaglandin E receptor antagonist ONO-8711 can exert a chemopreventive effect by inducing the apoptosis in breast tumors. In addition, the activation of retinoic acid receptor by utilizing natural or synthetic retinoids can inhibit the growth rates of nonsmall cell lung cancer cells. Recent findings indicate that peroxisome proliferator-activated receptors (PPARs) can modulate the proliferation and apoptosis manner in cancer cells of various species, which are considered to be novel targets for anticancer therapeutics. PPARs, including PPAR α, β, and γ subtypes, share a highly conserved characteristic feature of DNA binding motif that interacts with retinoid X receptor to form a heterodimer. This heterodimer thereby regulates the expression of related target genes by binding to peroxisome proliferator response elements which located at the downstream of specific DNA sequences. PPARs are ligand-activated transcription factors acting as the major modulators capable of regulating hormone homeostasis and inhibiting the inflammatory process. The oxidized lipids derived from oxLDL can activate PPARs to modulate the expression of inflammatory cytokines. Accumulating evidences indicate that PPAR γ serving as the cell cycle modulators can influence the proliferation and differentiation of cancer cells, and its ligand activation can significantly suppress the growth of human colon cancer cells and increase expression of carcinoembryonic antigen, as well as trigger the reversal of gene expression events. Similarly, PPAR γ activation by the administration of TZD can cause lipid accumulation in breast cancer cells, modulate the epithelial genes expression associated with cell differentiation, and inhibit the cancer cell growth rates. However, the role of PPAR α/γ agonist TZD18 in the growth and immune parameters of gastric cancer cells remains unclear.
In the present study, the growth rates and apoptotic gene expressions of gastric cancer cells following the exposure to PPARs agonist in vitro were analyzed so as to investigate the potential correlation between PPARs agonists and gastric cancer.
| > Materials and Methods|| |
Cell lines and reagents
Human gastric carcinoma cell line, MKN-45, (ATCC, Manassas, VA, USA) was cultured in RPMI-1640 medium (Invitrogen, New York, USA) supplemented with 10% fetal bovine serum. MKN-45 cell was maintained in a humidified incubator containing 5% CO2 at 37°C. TZD18 was kindly provided by Merck (NJ, USA).
3-(4, 5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide assay
MKN-45 Cell was seeded into 96-well plates and cultured for 12 h. Then, the medium was removed and replaced with fresh medium containing varying concentrations of TZD18 (12.5, 25, 50 μm) or solvent (dimethyl sulfoxide) alone. The chemosensitivity was assessed using a standard 3-(4, 5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay following a continuous 24, 48, and 72 h and exposure, respectively. The effects of TZD18 on cell cycle distribution on different time points and concentrations were determined by flow cytometry with propidium iodide (PI) staining.
The percentages of apoptotic cells were determined by flow cytometry as described previously. Briefly, cells were harvested and fixed in 70% ice-cold ethanol overnight at −20°C. After washing with phosphate-buffered saline (PBS), the cells were centrifuged and stained for 30 min in PBS containing PI (50 μg/ml; Sigma) and RNase A (100 μg/ml; Sigma, CA, USA). About 106 cells were measured and analyzed using a FACScan flow cytometer (Becton–Dickinson).
The subcellular localization of cytochrome c was examined using an antibody against cytochrome c. Cells were seeded on glass coverslips placed in 24 wells for 18 h and then incubated with fresh medium containing varying concentrations of TZD18 (10, 50 μm). After 36 h incubation, cells were fixed in 4% paraformaldehyde and then permeabilized in 1% triton X-100 for 15 min. After washing with PBS, cells were blocked with 2% bovine serum albumin for 30 min. Cells were incubated with mouse anti-cytoc antibody (1:100; Merck, NJ, USA) for 2 h, then incubated with a secondary antibody conjugated with fluorescein isothiocyanate (1:100; Pierce) for 1 h. After that, cells were stained with 6-diamidino-2-phenylindole for 8 min, washed with PBS, and then observed under a fluorescence microscope (Leica, Wetzlar, Germany).
Measurement of mitochondrial membrane potential
JC-1 was used to evaluate the alteration of mitochondrial membrane potential. Following the treatments with PPARs agonists, cells were washed with fresh culture medium and incubated with JC-1 dye (10 μg/ml) for 20 min. The percentages of cells with mitochondrial membrane potential (MMP) level were quantified by flow cytometry. Briefly, cells were collected and stained with 10 μg/ml JC-1 solution for 15 min. The cells were centrifuged at 500× g for 5 min and resuspended in 0.5 ml PBS for further flow cytometry analysis.
Western blotting assay
Protein extract was prepared and subjected to Western blotting assay as described previously. The protein concentration was determined using BCA kit. The proteins were electrophoresed by sodium dodecyl sulfate polyacrylamide gel electrophoresis containing 5% stacking gel and 12% separating gel. The proteins were electrically transferred to polyvinylidene fluoride membranes by using a semi-dry apparatus (Bio-Rad, CA, USA). After that, the membranes were incubated with mouse anti-Bcl2, anti-Bax, and anti-p27kip1 primary antibodies (Abcam, UK) for 2 h at room temperature and then were incubated with a secondary antibody conjugated with peroxidase-conjugated secondary antibodies for 1 h. The band on the membranes was visualized using ECL detection system ( Amersham Pharmacia Biotech, Germany). Anti-β-actin primary body (Abcam, UK) was used as an internal control. The gray-scale analysis for the protein expression was measured by ImageJ program. The experiments were performed in triplicate.
The data analysis was measured using SPSS 18 (SPSS Inc, Chicago, IL, USA) analysis program and represented as means ± standard deviation. All of the experimental data analyses were subjected to one-way analysis of variance (one-way ANOVA). In the further analysis of Duncan's Multiple Range Test, only if the level of P < 0.05, the differences were considered statistically significant.
| > Results|| |
Determination of MKN-45 cell proliferation rates
To investigate whether TZD18 could in reduce the proliferation rates of cancer cells, MTT assay against various human cancer cell lines were performed. As shown in [Figure 1], the proliferation rates in the control group maintained stable around 91.3%. In contrast, the proliferation rates in the treatment groups were consistently lower than that of the control group following TZD18 exposure. The proliferation rates of MKN-45 cells treated with 10 μm TZD18 showed a slight decrease, from 84.3% at 12 h postexposure to 69.8% at 72 h postexposure. A significant decrease of MKN-45 cell proliferation rates from 75.3% at 12 h postexposure to 45.3% at 72 h postexposure was observed in the treatment group with 50 μm TZD18. As illustrated in supplement [Figure 1], flow cytometry demonstrated that TZD18 significantly induced cell cycle arrest at G0/G1 phase with the increase in concentration. In conclusion, the results argued that TZD18 could in reduce the proliferation rates of MKN-45 cells.
|Figure 1: Effect of TZD18 on cellular viability in vitro. Human gastric carcinoma cell line (MKN-45) was treated with TZD18 at different doses for 24, 48, and 72 h. The viability was determined by the 3-(4, 5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide assay and the values are expressed as the mean ± standard deviation. Significantly different from control groups at P < 0.05|
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TZD18 induced the apoptotic process in MKN-45 cell
To characterize the mechanism involved in TZD18-induced cells death, we assessed if the effect of TZD18 on MKN-45 cells was due to apoptosis. Stained nuclei were observed under a fluorescent microscope using a blue filter, and condensed chromatin and nuclear shrivel were found [Figure 2]a. In [Figure 2]b, the apoptotic ratios in the control group maintained stable at approximately 6.1%. In contrast, the MKN-45 cells treated with 12.5 μm TZD18 showed a slight increase of apoptotic ratios from 8.2% at 24 h postexposure to 18.5% at 72 h postexposure. A significant increase of apoptotic ratios was observed at 24 h and exposure to 50 μm TZD18 and gradually increased to 47.1% following 72 h postexposure. In [Figure 2]c, the apoptosis percentage of cells staining with Annexin V-FITC and PI. The results were shown from one of three experiments with similar results. To sum up, the effect of reduced proliferation rates for TZD18 on MKN-45 cells was due to apoptosis.
|Figure 2: TZD18 could induce cell apoptosis in MKN-45 cells. (a) Cells were treated with 50 μM TZD18 for 72 h, fixed, stained with 4, 6-diamidino-2-phenylindole and stained nuclei, and observed under a fluorescent microscope. (b) Cells treated with 0, 12.5, 25, and 50 μM TZD18 for 24, 48, and 72 h were assessed for apoptosis by staining with Annexin V-FITC and propidium iodide. (c) The apoptosis percentage of cells treated with 0, 12.5, 25, and 50 μM TZD18 for 24, 48, and 72 h were assessed for apoptosis by staining with Annexin V-FITC and propidium iodide. *P < 0.05, compared with control|
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Regulation of caspase activities following the TZD18 exposure
Caspase activation, which contains caspase-3, caspase-8, and caspase-9, is known as a major step in apoptosis. To further identify the mechanism of TZD18-induced proliferation inhibition, expression of these caspases was tested. In [Figure 3]a, the activities of caspase-3 increased slightly after 72 h and exposure to 12.5 μm TZD18 and continuously increased to a peak level at 50 μm postexposure with the highest value of 3.1-fold greater than that of the control (P < 0.05) [Figure 3]b. The expression of caspase-8 treated with 12.5 μm TZD18 exhibited a significant increase at the beginning (72 h) and reached a peak level at 50 μm postexposure with the highest value of 4.3-fold greater than that of the control (P < 0.05). Similarly, the expression of caspase-9 treated with 50 μm TZD18 exhibited a significant increase with the highest value of 3.8-fold greater than that of the treated with 12.5 μm (P < 0.05). In all, our results indicated that TZD18-induced proliferation inhibition is by apoptosis and caspases are enrolled in this process.
|Figure 3: Regulation of caspase activities following TZD18 exposure. (a) The MKN-45 cell was treated with 12.5 and 50 μM TZD18 for 72 h lysed using RIPA/MPER and 40 μg of cell lysates were immunoblotted for caspase-3, caspase-8, and caspase-9 protein levels. β-actin was used to determine protein loading. (b) Data were represented as mean ± standard deviation of three separate experiments. Significant differences from control were indicated, *P < 0.05|
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TZD18 exposure modulated the mitochondrial membrane potential and the release of cytochrome c in MKN-45 cell
To reveal whether mitochondrial dysfunction participated in the induction of apoptosis or even played a pivotal role in the apoptotic process, mitochondrial membrane potential damage was evaluated using JC-1 reagent using flow cytometric analysis and mitochondrial and cytochrome c translocation were observed with fluorescent photomicrograph. In [Figure 4]a, the percentage of cells in 12.5 μm TZD18 treatment group with depolarized MMP increased from 12.3% at 24 h postexposure to 26.4% at 48 h postexposure, whereas the cells treated with 50 μm TZD18 showed a sharp increase of MMP depolarization at 24 h postexposure and continuously increased to 34.5% at 72 h postexposure [Figure 4]b. In [Figure 4]c, the distribution pattern of cytochrome c was colocalized with the mitochondria in the control cells. In contrast, a sharp increase of cytochrome c release was observed following 72 h and exposure from 12.5 to 50 μm TZD18. Together, the results showed that mitochondrial dysfunction was indeed participated in the induction of apoptosis and played a pivotal role in the apoptotic process.
|Figure 4: TZD18 exposure modulated the mitochondrial membrane potential and the release of cytochrome c in MKN-45 cell. (a) Mitochondrial membrane potential damage was evaluated using JC-1 reagent using flow cytometric analysis. (b) The results are presented as means ± standard deviation of three independent experiments (*P < 0.05). (c) Fluorescent photomicrograph of mitochondrial (Green) and cytochrome c (Red) translocation treated by 12.5 and 50 μM TZD18 for 72 h|
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Effects of TZD18 exposure on the expression of p27kip1 Bcl-2, Bax
To investigate the effect of TZD18 on the apoptosis-related protein expression, the cells were exposed to 12.5 or 50 μm TZD18 for 72 h. In [Figure 5], the expression of p27kip1 increased significantly in the TZD18 treatment group following 72 h postexposure by comparing with that of the control. In addition, Bax expression increased dose dependently following 72 h and exposure to various doses of TZD18, while the Bcl-2 expression decreased. The result further pointed out that TZD18 plays a role on the proliferation rates of MKN-45 cells by apoptosis.
|Figure 5: Effects of TZD18 exposure on the expression of p27kip1, Bcl-2, and Bax. (a) The expression of p27kip1, Bcl-2, and Bax was analyzed by Western blot with the loading control of β-actin. (b) Significant differences from control were indicated, *P < 0.05|
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| > Discussion|| |
PPAR γ is a ligand-dependent transcription factor that can modulate a variety of physiological processes including regulation of fat cell development and maintenance of glucose homeostasis., In cancer study, recent findings indicate that PPAR γ is playing an important role in the antitumor processes as a negative modulator capable of inducing the apoptosis by increasing the expression of p27kip1 protein in the gastric cancer cell line through a p53-dependent mechanism. In addition, the administration of Troglitazone can inhibit cell growth and induce differentiation markers in colon cancer cell lines. TZD18 is a newly synthesized dual agonist for PPAR α/γ that can exhibit anti-proliferative effect by activating PPAR α and γ. Previous studies indicate that the administration of TZD18 can significantly inhibit cell growth and induce apoptosis process in T98G cells. However, there are little data on the parameter changes of MKD-45 cells following exposure to TZD18 treatment. In this study, we investigated the effect of TZD18 exposure on the MKN-45 cell. Following the exposure to TZD18 treatment, the growth of MKN-45 cells was inhibited by TZD18 exposure in a dose-dependent manner, implying that TZD18 exposure has anti-proliferative effect on MKN-45 cells.
Evidence is emerging that the agonists of synthetic TZD class can cause G1 arrest in colon cancer cells by increasing the expression level of p21Waf-1, Drg-1, and E-cadherin, which are similar to other cancer cells exposed to TZD including pancreatic cancer cells and breast cancer cells. In general, the cell cycle withdrawal in cancer cells by TZDs may be related to the increased level of CDK inhibitors such as p27kip1. In this study, a sharp increase of p27kip1 expression was observed following TZD18 exposure. Previous studies indicate that the T98G cell cycle withdrawn may be associated with upregulation of p27kip1, implying that TZD18 exposure may possibly cause the induced G1 arrest in MKN-45 cells.
Indeed, cell cycle arrest is one of the prerequisite conditions for apoptosis. To determine the underlying mechanisms of the growth inhibitory effect by TZD18 treatment, we also investigated the induced apoptosis. It is found that TZD18 exposure could induce apoptosis in MKN-45 cells in a dose-dependent manner. Caspase is playing an important role in the apoptotic cell death as a class of cysteine proteases capable of cleaving the critical cellular substrates such as lamins and poly (ADP-ribose) polymerase, thus triggering the significant changes of apoptotic process. Cells undergoing apoptosis following the activated apoptotic pathway can execute programed cell death by activating a hierarchy of caspases. Besides, caspase-3 activated by caspase-9 is essential to dismantle the cell function and form the apoptotic bodies., Previous studies indicate that the chemical exposure can cause the apoptotic process by increasing the expression of caspase-3 and caspase-9 and modulating the mitochondrial membrane potential. A variety of major events in apoptosis may also link to different signals that converge on mitochondria to trigger or block these events including the loss of mitochondrial membrane potential, release of cytochrome c, change in electron transport, and the involvement of pro- and anti-apoptotic Bcl-2 family proteins. In this study, we found that the caspase-3 and caspase-9 activity increased significantly along with a sharp increase of MMP and cytochrome c release, indicating that TZD18-induced apoptosis may be associated with the caspase activation. In addition, among dozens of apoptotic-related molecules, Bcl-2 is playing an important role in apoptosis by inhibiting the programed cell death process, whereas Bax is a member of the proapoptotic proteins that can be regulated by chemotherapeutic drugs through a p53-dependent mechanism. Previous studies indicate that PPAR α/γ ligand TZD18 can induce a remarkable apoptosis in leukemia cell lines by increasing the expression level of Bax. In addition, the administration of TZD18 can upregulate the Bax expression but decrease Bcl-2 expression in human glioblastoma T98G cells. In this study, we found that TZD18 exposure inhibited the expression of Bcl-2 at the protein level, while an upregulation of Bax expression was observed, suggesting that the apoptotic mechanism for the administration of TZD18 mediated apoptosis process by increasing Bax expression but decreasing Bcl-2 expression.
| > Conclusion|| |
The current study showed that TZD18 inhibited cell growth and induced apoptosis in MKN-45 cells, which linked to activation of Bax, caspase-3, and caspase-9 along with a sharp increase of MMP and cytochrome c release but downregulation of Bcl-2 expression, suggesting that TZD18 might have a therapeutic role in the treatment of human gastric cancer. Further studies using other cells as well as potential synergy with other agents should now be pursued.
Financial support and sponsorship
This research was funded by the National Natural Science Foundation of Henan (No.2015GGJS-286, 17A180010) and China Postdoctoral Science Foundation Funded Project (2016M590143).
Conflicts of interest
There are no conflicts of interest.
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