Cancer Cell Biology
Growth arrest by troglitazone is mediated by p27Kip1 accumulation, which results from dual inhibition of proteasome activity and Skp2 expression in human hepatocellular carcinoma cells
Version of Record online: 7 OCT 2003
Copyright © 2003 Wiley-Liss, Inc.
International Journal of Cancer
Volume 108, Issue 1, pages 41–46, 1 January 2004
How to Cite
Motomura, W., Takahashi, N., Nagamine, M., Sawamukai, M., Tanno, S., Kohgo, Y. and Okumura, T. (2004), Growth arrest by troglitazone is mediated by p27Kip1 accumulation, which results from dual inhibition of proteasome activity and Skp2 expression in human hepatocellular carcinoma cells. Int. J. Cancer, 108: 41–46. doi: 10.1002/ijc.11561
- Issue online: 7 NOV 2003
- Version of Record online: 7 OCT 2003
- Manuscript Accepted: 4 AUG 2003
- Manuscript Revised: 30 JUN 2003
- Manuscript Received: 1 APR 2003
- growth arrest;
- hepatocellular carcinoma
In our study, we examined whether human hepatocellular carcinoma (HCC) expresses peroxisome proliferator-activated receptor γ (PPARγ) and the effects of PPAR γ activation by its selective ligands on cell growth and cell invasion in HCC cells. RT-PCR and Western blot analysis revealed that HCC-derived cell lines, HepG2 and HLF, express PPARγ mRNA and protein. Luciferase assay in HLF cells showed that troglitazone, a selective ligand for PPAR γ, transactivated the transcription of a peroxisome proliferator response element-driven promoter in a dose-dependent manner, suggesting that the expressed PPARγ functions as a transcriptional factor. Not only troglitazone but pioglitazone dose-dependently inhibited cell growth in HepG2 and HLF cells. Invasion assay using a transwell chamber demonstrated that troglitazone also inhibited cell invasion in HCC cells. To examine the mechanism of the troglitazone-induced growth inhibition, we determined p27Kip1, a cyclin dependent kinase inhibitor, expression by Western blot analysis in troglitazone-treated HLF cells. Troglitazone increased p27Kip1 in time- and dose-dependent manners, suggesting that p27Kip1 may be involved in the growth inhibition by troglitazone in HLF cells. To further examine the mechanism of the troglitazone-induced p27Kip1 protein accumulation, 2 major systems for regulation of p27Kip1 protein, proteasome activity and Skp2, an F-box protein that targets p27Kip1 for degradation, were evaluated. Troglitazone potently inhibited proteasome activity and decreased Skp2 protein levels. All these results suggest that human HCC cells express functional PPAR γ and PPARγ activation resulted in growth inhibition. The growth inhibition was mediated by p27Kip1 accumulation, which is induced by both inhibition of ubiquitylation of p27Kip1 and reduction of degradation activity of p27Kip1 by proteasome. © 2003 Wiley-Liss, Inc.
Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily that includes receptors for steroids, thyroid hormone, vitamin D and retinoic acid.1 PPARγ is expressed at high levels in adipose tissue and functions as a key molecule of adipocyte differentiation.2, 3 In addition to adipose tissue, PPARγ expression is detected in a wide variety of tumor cells.4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 In the tumor cells, PPARγ activation by its high affinity ligands could inhibit cell growth. Thus PPARγ is involved in not only lipid metabolism but also cellular proliferation in cancer cells. It is therefore suggested that PPARγ is considered as a possible molecular target for cancer treatment.
Although increasing evidence have established that PPARγ activation induces growth arrest in cancer cells, the molecular mechanism of the growth inhibition by PPARγ ligands had not been well understood. With regard to this point, we have very recently demonstrated that p27Kip1, a cyclin-dependent kinase inhibitor (CDKI),16, 17 may be a key molecule in the cell growth inhibition by troglitazone in human pancreatic cancer cells.14 We do not know however the relationship between PPARγ activation and up-regulation of p27Kip1 protein expression. In our study, we examined whether human HCC cells expresses PPARγ, the effect of PPARγ activation on cell proliferation and cell motility and the mechanism of the accumulation of p27Kip1that plays a vital role in the growth inhibition.
MATERIAL AND METHODS
Two HCC-derived cell lines designated HepG2 and HLF were used. HepG2 cells18 were obtained from the American Type Culture Collection (Rockville, MD) and were cultured in Dulbecco's modified Eagles medium (GIBCO, Grand Island, NY) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2.5 μg/ml amphotericine and 10% fetal bovine serum. HLF19 was obtained from Japanese Cancer Research Resources Bank (Tsukuba, Japan). HLF cells were cultured in RPMI-1640 medium (GIBCO, Grand Island, NY) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2.5 μg/ml amphotericine and 10% fetal bovine serum. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air.
Reagents and Treatments
Troglitazone and pioglitazone were kindly provided from Sankyo Pharmaceutical Co. (Tokyo, Japan) and Takeda Chemical Industries (Tokyo, Japan), respectively. These thiazolidinediones were dissolved in dimethyl sulfoxide (DMSO) with a final concentration of DMSO 0.05 % in the culture medium.
Total RNA was extracted from cultured cells using a modified version of the acid guanidinium thiocyanate/phenol/chloroform method employing a single reagent (RNA-STAT 60, TelTest, Inc., Friendswood, TX).20, 21 Samples were dissolved with diethyl pyrocarbonate-treated water (RNase-free). To remove contaminating genomic DNA, the RNA was treated with 10 μl of RQ1, RNase-free DNase (Promega, Madison, WI), 0.5 μl of RNase inhibitor (Takara Shuzou Co., Otsu, Shiga, Japan) and 10 μl of 10 × DNase buffer (400 mM Tris-HCl at pH 7.9, 100 mM NaCl, 60 mM MgCl2 and 100 mM CaCl2) in a final volume to 100 μl for 30 min at 37°C. RNA samples were purified by phenol-chloroform extraction and isopropanol precipitation. The resultant RNA samples were quantified using spectrophotometer at a wave length of 260 nm. The integrity of the isolated RNA samples were analyzed electrophoretically on an agarose gel, followed by staining in ethidium bromide.
Reverse transcription PCR (RT-PCR)
An aliquot of 1 μg of total RNA from each sample was reverse transcribed to cDNA using First-Strand cDNA synthesis Kit (Pharmacia LKB Biotechnology, Uppsala, Sweden) according to manufacturer's instructions with oligo (dT) primer. For detection of the human PPARγ mRNA, a combination of a sense primer of 5′-TCTCTCCGTAATGGAAGACC-3′ and an antisense primer of 5′-GCATTATGAGACATCCCCAC-3′ was used as described previously.22 The amplification was carried out in a 100 μl mixture containing 1 μl of the above cDNA product (corresponding to cDNA synthesized from 67 ng of total RNA), 0.4 μM each of the sense and antisense primers, 10 mM Tris HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTPs and 2.5 units of Taq DNA polymerase (Takara Shuzou Co., Otsu, Shiga, Japan). The reaction conditions were as follows: initial denaturation at 95°C for 2 min and 40 cycles of amplification (95°C for 40 sec, 55°C for 50 sec and 72 °C for 50 sec), followed by a final extension step of 7 min at 72°C. The PCR reaction products were separated electrophoretically in a 2 % agarose gel and stained with ethidium bromide.
PPARγ protein expression detected by Western blot analysis
Total proteins were extracted from HepG2 and HLF cells. Protein concentrations were measured using Bio-Rad Protein Assay Reagent (Bio-Rad Lab., Richmond, CA) following the manufacturer's suggested procedure. Fifty micrograms of protein was separated by 10 % SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose membrane (Amersham Life Science, Inc., Piscataway, NJ), blocked overnight in PBS with 5 % skim milk at 4°C, subsequently reacted with primarily polyclonal antibody against human PPARγ (Carbiochem, San Diego, CA) and washed. After reaction with horseradish peroxidase-conjugated anti-rabbit IgG, immune complexes were visualized by using the ECL detection reagents (Amersham, Buckinghamshire, UK) following the manufacturer's suggested procedure. Simultaneously, nonimmune rabbit IgG (Pharmingen, San Diego, CA) was used for control.
Transfections and luciferase assays
Transfections and luciferase assays were performed according to a previous report.14 HLF cells were seeded at a concentration of 1 × 105 cells/35 mm dish and transfected with the plasmids 24 hr after having been transferred to fresh media. Transfection was done by using Lipofectamine reagent (Gibco BRL) mixed with 2 μg of acyl CoA oxidase promoter-luciferase plasmid (kindly donated by Dr. Osumi)23 and 0.2 μg of pRL-SV40 (Promega, Madison, WI, USA) for 3 hr. The transfection mix was replaced by complete media with DMSO, 0.1, 1, 10 or 100 μM troglitazone and further incubated for 12 hr. The cells were lysed with 1 × luciferase lysis buffer (Toyo Inc., Tokyo, Japan). Luciferase activity was measured using the PicaGene reagent kit (Toyo Inc., Tokyo, Japan) in a luminometer (MiniLumat, Berthold, Widbad, Germany). The enzyme activity was normalized for efficiency of transfection, on the basis of sea pansy luciferase activity, and relative values were determined. Transfection experiments were carried out fo4ur times independently, and the average values were calculated.
Cell growth assay
To evaluate the effect of thiazolidinediones on cell growth, cells were seeded on a 96-well cell culture cluster (Corning Inc., Corning, NY) at a concentration of 2 × 104/well in a volumes of 100 μl. Twenty-four hour later, each well was incubated with troglitazone or pioglitazone at several concentrations for 0, 24 or 48 hr. Cell numbers were measured colorimetrically using the Cell Counting Kit (Dojindo, Kumamoto, Japan) by ImmunoMini NJ-2300 (NJ InterMed, Tokyo, Japan) at a test wavelength of 450 nm. This assay is based on the cleavage of the 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) by mitochondrial dehydrogenase in viable cells.24
Invasion assay in HLF was performed using transwell chambers (Costar, Cambridge, MA) with 8 mm diameter, tissue culture-treated filters with 8 μm pores according to the method of Repesh25 with some modifications. Tumor cells (5 × 105/ml) were suspended in RPMI supplemented with 0.1 % bovine serum albumin, and the cell suspensions (100 μl) were then placed into the upper compartment of a transwell chamber. RPMI and 15 μg/ml dose of fibronectin were then placed into the lower compartment. After incubation for 10 hr, cells that penetrated through the filters were counted. Each filter was fixed with 3 % paraformaldehyde in Dulbeco's phosphate-buffered saline and stained in Giemsa solution. After the cells attached to the upper side of the filter were removed by wiping with a cotton swab, the cells attached to the lower side of the filter were counted using a microscope. The total number of cells in the lower transwell compartment and on the lower side of filter were determined, and chemotaxis was expressed as the number of cells penetrating through the filter per 5 × 104 cells added to the upper compartment.
Protein expression p27Kip1 and Skp2 detected by Western blot analysis
Effect of troglitazone on the expression of p27Kip1 and Skp2 in HLF cells was studied by Western blot analysis. HLF cells were treated with several doses of troglitazone and total proteins were extracted from HLF cells at several time points. Protein concentrations were measured using Bio-Rad Protein Assay Reagent (Bio-Rad Lab., Richmond, CA) following the manufacturer's suggested procedure. Fifty micrograms of protein was separated by 5–20 % SDS-PAGE (Ready Gels J, Bio-Rad Richmond CA). After electrophoresis, the proteins were transferred to nitrocellulose membrane (Amersham Life Science, Inc., Piscataway, NJ), blocked overnight in PBS with 10 % skim milk at 4°C, subsequently reacted with primarily polyclonal antibody against human p27Kip1 (Santa Cruz Lab., Santa Cruz, CA) or polyclonal antibody against human Skp2 (Zymed Lab, Inc., San Francisco, CA) and washed. After reaction with horseradish peroxidase-conjugated anti-goat IgG, immune complexes were visualized by using the ECL detection reagents (Amersham, Buckinghamshire, UK) following the manufacturer's suggested procedure. Simultaneously, normal goat IgG was used for control. Blots were reprobed with antibodies to actin (Santa Cruz Lab.) for comparison of results.
The proteasome assays were performed after treatment of HLF cells with troglitazone for 48 hr, and the medium was removed and the cells were washed several times before they were lysed. To measure the proteasome chymotrypsin peptidase activity, 10 μl of cellular extract (100 μg, prepared by brief sonication of cells and fractionation at 15,000g) was diluted in a cuvette containing 2 ml of 20 mM Hepes, 0.5 M EDTA, pH 8, and 0.035 % SDS. The cell extracts contain a mixture of 26S proteasome. The above mixture was incubated at 37°C before the addition of the fluorogenic substrate, 10 μM succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. Substrate hydrolysis was measured by continuous monitoring for fluorescence (emission at 460 nm, excitation at 380 nM) of the liberated 7-amido-4-methylcoumarin for 750 sec as described.26
The results are expressed as mean ± SEM. Statistical analysis was performed by repeated measures ANOVA and subsequent Fisher's LSD test. p < 0.05 was considered statistically significant.
We first examined if PPARγ gene is expressed in human HCC cell lines, HepG2 and HLF. Figure 1a (lane 1 and 2) illustrates the representative result of PPARγ gene expression by RT-PCR. PPARγ mRNA is detected in both HepG2 and HLF cells. Figure 1b demonstrates a representative result of Western blot analysis. PPARγ protein was expressed in HepG2 and HLF cells, indicating that human HCC cells express PPARγ.
Next, we examined if the PPARγ expressed in human HCC cells functions as a transcriptional factor as shown in different type of cells such as adipocytes, and colon, gastric and pancreatic cancer cells.2, 4, 5, 11, 14 HLF cells were transfected with an acyl-CoA oxidase promoter-luciferase reporter plasmid containing a peroxisome proliferator response element (PPRE). Treatment with troglitazone, a selective ligand for PPARγ27 for 12 hr increased the luciferase activity in the HLF cells in a dose-dependent fashion (Fig. 2).
The effect of PPARγ activation on cell growth in HepG2 and HLF was examined using WST-1 assay. HepG2 or HLF cells were continuously cultured in several doses of troglitazone or pioglitazone, specific ligands for PPARγ, and cell numbers were determined. Figure 3 illustrates the dose-response and time-course effects of troglitazone or pioglitazone on the number of HepG2 cells. Treatment with troglitazone significantly inhibited the growth of HepG2 cells in a dose-dependent fashion (Fig. 3a). Another thiazolidinedione, pioglitazone, similarly resulted in growth inhibition of HepG2 cells (Figure 3b). As shown in Figure 4, troglitazone (Fig. 4a) and pioglitazone (Fig. 4b) also inhibited the growth of HLF cells. The inhibitory action of thiazolidinediones on HLF cells was dose-dependent. Invasion assay using a transwell chamber revealed that troglitazone inhibited cell invasion through a filter in a dose-dependent manner (Fig. 5).
To examine a role of p27Kip1 in the growth arrest induced by thiazolidinediones in HLF cells, p27Kip1 protein expression was evaluated by Western blot analysis. As clearly demonstrated in Figure 6, troglitazone increased the amount of p27Kip1 in HLF cells in time- and dose-dependent fashions, suggesting that p27Kip1 may be involved in the growth inhibition by troglitazone in HLF cells.
Next, we examined the effect of troglitazone on proteasome activity and showed that proteasome activity was dose-dependently inhibited by troglitazone (Fig. 7). To clarify whether troglitazone changes p27Kip1 ubiquitylation, Skp2 protein levels were analyzed by Western blotting. As shown in Figure 8, Skp2 protein expression was inhibited by troglitazone in dose- and time-dependent manners.
We first examined whether 2 established HCC cell lines, HepG2 and HLF, express PPARγ. The PPARγ mRNA and protein expression were detected by RT-PCR and Western blot analyses in these 2 cell lines, being in good agreement with previous reports.28 The transient transfection assays in HLF cells using a PPAR-responsive element cloned upstream of luciferase to determine whether the PPARγ expressed in HLF cells reveled that the expressed PPARγ is functional as a transcriptional factor.
Recent evidence has demonstrated that PPARγ activation by thiazolidinediones such as troglitazone induced inhibition of a wide variety of cancer cells,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 suggesting that activation of PPARγ by thiazolidinediones down-regulates cell growth. The evidence obtained in our study that application of troglitazone or pioglitazone significantly inhibited the cell growth in HepG2 and HLF cells suggests that PPARγ activation by thiazolidinediones could prevent cell growth in HCC cells, further supporting previous results.28
Invasion assay using transwell chamber revealed that troglitazone significantly suppressed HLF cell invasion, suggesting for the first time that cancer cell motile activity is inhibited by PPARγ activation. Since cell invasive activity plays an important role in tumor cell progression,29, 30, 31 the troglitazone-induced suppression of cell-invasive activity may be favorable. PPARγ ligand-induced inhibition of cell invasive activity in addition to cell proliferation in HLF cells may further support our speculation that PPARγ ligand may be useful for the treatment of human HCC.
The troglitazone-induced growth arrest as seen in the present study may explain a mechanism by which NSIADs evoke growth inhibition in HCC cells. Rahman et al.32 have recently demonstrated that sulindac, a nonsteroidal anti-inflammatory drug (NSAID), exhibits a potent antiproliferative effect in human HCC cells. Although the precise mechanism of this pharmacological action of NSAIDs on HCC cells is not fully clarified, one possible explanation is that NSAIDs act as a cyclooxygenase inhibitor to inhibit the growth of HCC cells. However, other mechanisms of inhibiting HCC cell growth cannot be excluded. According to a previous report,33 NSAIDs are known to be weak agonists of PPARγ. We may therefore speculate that PPARγ activation may contribute, at least in part, to the growth inhibition in HCC cells by NSAIDs.
Earlier investigators have demonstrated the different effects on cell behavior of troglitazone and pioglitazone.34, 35 The present evidence was largely obtained from experiments with troglitazone. Further studies are needed to clarify whether other thiazolidinediones have the same effects as troglitazone. In addition, it has been demonstrated that troglitazone induces not only cell growth arrest but apoptosis in cancer cells. In human hepatoma cells, a number of articles reported that troglitazone induced apoptosis in hepatoma cells.36, 37 Although the precise mechanism of induction of apoptosis by troglitazone is not clarified in hepatoma cells, we have demonstrated very recently that the troglitazone-induced apoptosis in human gastric cancer cells is mediated through a p53-dependent pathway.38 The same mechanism might mediate the troglitazone-induced apoptosis in hepatoma cells. In this regard, further study is needed.
With regard to the mechanism by which PPARγ activation induces growth inhibition, we have very recently demonstrated that p27Kip1, a CDKI, may be a key molecule that is implicated in the cell growth arrest by troglitazone in human pancreatic cancer cells because troglitazone increased the level of p27Kip1 protein but not other CDKI, p21 or p18, protein and the inhibition of cell proliferation by troglitazone was not observed in cells transfected with an antisense oligonucleotide against p27Kip1.14 In addition, p27Kip1 accumulation was observed in gastric cancer cells as shown in our previous reports26, 38 and in HCC cells in our study, suggesting that the increased level of p27Kip1 may be a common mechanism by which PPARγ activation induces the inhibition of cell growth in a wide variety of cancer cells.
Although we do not know completely the mechanism of accumulation of p27Kip1 by troglitazone, it has been reported that a ubiquitin-proteasome pathway is implicated in one of the posttranslational mechanisms of p27Kip1 regulation.22, 23, 24 Based upon this evidence, we have recently shown that troglitazone inhibited proteasome activity in human gastric cancer cells.26 Since a proteasome inhibitor, lactacystin, inhibited proteasome activity, and increased the protein level of p27Kip1 as well as troglitazone does in human gastric cancer cells, we hypothesized that troglitazone inhibits proteasome activity to accumulate p27Kip1 protein in gastric cancer cells.26 As demonstrated in the present study, proteasome activity was also inhibited in human HCC cells, further supporting that p27Kip1 protein accumulation results from the inhibition of proteasome by troglitazone. From another point of view, we examined whether ubiquitylation of p27Kip1 is modified by troglitazone. Skp2 is a key enzyme for p27Kip1 ubiquitylation as a ubiqutin ligaze.39 The present study illustrated that Skp2 protein expression was potently inhibited by troglitazone. This evidence suggests that troglitazone suppressed p27Kip1 ubiquitylation. We therefore speculated that the decreased activity of ubiquitylation of p27Kip1 by troglitazone plays a role in the accumulation of p27Kip1 by preventing degradation of p27Kip1 from proteasome. All these results suggest that p27Kip1 accumulation by troglitazone is induced by dual mechanisms: inhibition of proteasome activity and inhibition of ubiquitylation of p27Kip1.
- 20Molecular cloning. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989., , .