Thyroid hormone (triiodothyronine, T3) regulates growth, development and differentiation. To examine the influence of T3 on hepatoma cell growth, thyroid receptor (TR)α1 or TRβ1 over-expressing HepG2 cell lines were used. Growth of the HepG2-TR stable cell line was inhibited by over 50% following treatment with T3. However, transforming growth factor (TGF)-β neutralizing antibody, but not the control antibody can reverse the cell growth inhibition effect of T3. Flow cytometric analysis indicated that the growth inhibition was apparent at the transition point between the G1 and S phases of the cell cycle. The expression of major cell cycle regulators was used to provide further evidence for the growth inhibition. Cyclin-dependent kinase 2 (cdk2) and cyclin E were down-regulated in HepG2-TR cells. Moreover, p21 protein or mRNA levels were up-regulated by around 5-fold or 7.3-fold respectively following T3 treatment. Furthermore, phospho-retinoblastoma (ppRb) protein was down-regulated by T3. The expression of TGF-β was studied to delineate the repression mechanism. TGF-β was stimulated by T3 and its promoter activity was enhanced six- to eight-fold by T3. Furthermore, both T3 and TGF-β repressed the expression of cdk2, cyclin E and ppRb. On the other hand, TGF-β neutralizing but not control antibody blocked the repression of cdk2, cyclin E and ppRb by T3. These results demonstrated that T3 might play a key role in liver tumor cell proliferation.
The thyroid hormone, 3,3′,5-triiodo-l-thyronine (T3), mediates numerous physiological processes, including embryonic development, cellular differentiation, metabolism and the regulation of cell proliferation (Hulbert 2000, Aranda & Pascual 2001). T3 controls these processes in most organs. The effects of T3 are mediated by nuclear thyroid hormone receptors (TRs). Moreover, TRs bind to the thyroid hormone response elements (TREs) located upstream from the promoters of target genes to regulate their expression transcriptionally (Hulbert 2000, Aranda & Pascual 2001). The nature of the transcriptional response is determined by cell type, promoter context, and hormone status (Hulbert 2000, Aranda & Pascual 2001). In most cases, TRs are transcriptional repressors without their cognate hormone (T3 or thyroxine (T4)) and are turned into activators by ligand binding (Hulbert 2000, Aranda & Pascual 2001).
Two main types of TRs have been identified, termed TRα and TRβ, which are encoded on human chromosomes 17 and 3 respectively (Cheng 2000, Aranda & Pascual 2001). Transcripts of each of these genes undergo alternative promoter choice for generating both the TRα1 and α2 and the TRβ1 and β2 receptor isoforms (Cheng 2000, Hulbert 2000, Aranda & Pascual 2001).
Regarding previously published results (Lin et al. 2004), transforming growth factor-beta (TGF-β) was stimulated by T3 at the mRNA level. TGF-β regulates cell growth and proliferation, and has been shown to block the growth of numerous cell types (De Caestecker 2004). The TGF-β receptor includes type 1 and type 2 subunits. These subunits comprise serine-threonine kinases that signal through the smad family of transcriptional regulators. T3/T4 have been shown to stimulate the proliferation of eukaryotic cells (Barrera-Hernandez et al. 1999, Aranda & Pascual 2001). Several studies demonstrated that cyclin D1 induction is an early event in T3-induced hepatocyte proliferation (Pibiri et al. 2001, Alisi et al. 2004). These previous studies indicate that this cyclin may be a common target responsible for mitogenic activity of ligands of nuclear receptors. However, the influence of T3 on human liver tumor cell proliferation is currently unknown although a similar observation has been reported in rats (Ledda-Columbano et al. 2000).
The liver has long been recognized as a target organ for thyroid hormones. In fact, Chamba et al.(1996) reported that roughly equal quantities of TRα1 and TRβ1 protein occur in human hepatocytes (Chamba et al. 1996). HepG2 is a well-differentiated hepatocellular carcinoma cell line without detectable TR protein expression. However, it secretes all 15 plasma proteins and preserves numerous liver-specific functions and thus can serve as an in vitro model (Chang et al. 1983). Consequently, the HepG2 cell line provides a useful model system for studying the influence of T3 on the proliferation of liver tumor cells. The system was recently used to demonstrate that TGF-β is regulated by T3 (Lin et al. 2004). This work shows that T3 up-regulates the expression of TGF-β and subsequently suppresses liver tumor cell proliferation.
Materials and methods
Human hepatoma cell lines, HepG2-TRα1#1, HepG2-TRα1#2, HepG2-TRβ1 and HepG2-Neo, and rat pituitary tumor GC cells were routinely grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum. Three TR over-expressing lines, and the control cell line, HepG2-Neo, have been described previously (Lin et al. 2004). The serum was depleted of T3 (Td) as described by Samuels et al.(1979). Cells were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO2.
Flow cytometric analysis was performed as described by Fan et al.(1995). Briefly, cells were harvested via trypsinization and fixed in 75% ethanol for at least 24 h at 4 °C. The cells were then washed with PBS containing 1% BSA (Life Technologies, Inc., Rockville, MD, USA) and incubated with 100 μg/ml RNase A (Sigma, St Louis, MO, USA) and 50 μg/ml propidium iodide (Sigma) for 2 h at room temperature. Finally, the stained cells were analyzed on a FACS Calibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA).
Cell proliferation assay
Cells were plated on 6-cm dishes at 2 × 105cells/dish, with each sample being plated in triplicate. Cells were counted using the Coulter Counter ZM (Coulter Electronics Inc., Luton, Beds, UK).
Cell lysates were fractionated using SDS-polyacrylamide gel electrophoresis (PAGE) on a 10% gel, and the separated proteins were transferred to a nitrocellulose membrane (Pall Life Sciences, Ann Arbor, MI, USA) and subsequently visualized via chemiluminescence using an ECL detection kit (Amersham Inc., Piscataway, NJ, USA) as described previously (Shih et al. 2004). The antibodies used were rabbit polyclonal antibodies to cyclin E, and retinoblastoma (Rb) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse monoclonal antibody to cdk2 (1:1000 dilution in PBS) (Santa Cruz Biotechnology) or TGF-β (1:500–1000 dilution in PBS) (Serotec Ltd, Oxford, Oxon, UK). TGF-β1 was purchased from Pepro Techec (London, UK).
Northern blot analysis
Total RNA was extracted from the cells using TRIzol Reagent (Life Technologies) and equal amounts of total RNA (20 μg) were analyzed on a 1.2% agarose-formaldehyde gel as described previously (Lin et al. 2000, 2002). The separated RNA molecules were then transferred to a nylon membrane (Amersham) and subjected to northern blot analysis as described previously (Lin et al. 2004, Shih et al. 2004).
Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR)
Total RNA was extracted from cells using TRIzol, as described above. Subsequently, cDNA was synthesized using the Superscript II kit for RT-PCR (Life Technologies) as described previously (Shih et al. 2004).
Real-time Q-RT-PCR was performed in a 25-μl reaction mixture containing 50 nM forward and reverse primers, 1× Syber Green reaction mix (Applied Biosystems, Werrington, UK), and various quantities of template as described previously (Shih et al. 2004). Fluorescence emitted by Syber Green was detected using the ABI PRISM 7000 sequence detection system (Applied Biosystem), as described previously (Lin et al. 2004, Shih et al. 2004).
Cloning the TGF-β 5′-flanking region and promoter activity assay
Fragments of the TGF-β promoter (nucleotides −1362/+101) were amplified via PCR, according to the published nucleotide sequence (Kim et al. 1989a,b), and were then inserted into the pGL3 vector (Promega Corp., Madison, WI, USA). The sequence of the promoter construct was confirmed by automated DNA sequencing. To determine the influence of T3 on the transcriptional activity of the TGF-β promoter, HepG2-TRα1#1 cells (1 × 105 per 35-mm dish) were co-transfected, via a Lipofectamine protocol, using 3 μg pGL3 vector containing TGF-β promoter sequences (Invitrogen) as described previously (Shih et al. 2004).
T3 represses HepG2-TRα1 and HepG2-TRβ1 cell growth by prolonging the G0/G1 phase
The effect of T3 on cell proliferation was assayed in three HepG2 stable cell lines with high expression of TRα1 or TRβ1 as previously described (Lin et al. 2004). Figure 1 shows that without T3 cells proliferated roughly 2 to 3 times faster than those grown in media containing either 10 or 100 nM T3 in three TR stable lines. However, this T3-repressed effect was not observed in the control cell line, HepG2-Neo, which did not express TR (Fig. 1). Following T3 treatment, the doubling time for HepG2-TR stable lines increased from 1.03 ± 0.12 to 2.49 ± 0.31 days for TRα1#1 cells, and from 1.15 ± 0.21 to 2.64 ± 0.25 days for TRβ1 cells; thus T3 repressed the growth of HepG2-TR cells by around two- to three-fold. However, T3 did not influence the doubling time in HepG2-Neo cells (1.58 vs 1.67 days, 0 vs 100 nM T3). All data indicate that T3 significantly suppresses the growth of HepG2-TR but not HepG2-Neo cells.
To identify the phase of the cell cycle affected by T3, cell cycle distribution was assayed via flow cytometry. Table 1 illustrates that the addition of T3 increased the percentage of cells in the G0/G1 phases by roughly 1.2-, 1.4-, and 1.35-fold following 12, 24, and 48 h respectively. Simultaneously with the increase in cell number in the G0/G1 phases, the percentage of cells in the S phase was reduced in HepG2-TRα1#1 (Table 1). Similar results occurred in HepG2-TRβ1 (Table 1) and HepG2-TRα1#2 cells (data not shown).
T3 represses the protein expression levels of cdk2 and cyclin E
The major kinase associated with cyclin E in human cells is cdk2. The formation of the cyclin E–cdk2 complex is an important step in the biochemical pathway that controls cell proliferation during G1. Additionally, cyclin E is one of the key regulators of the G1/S transition in the cell cycle. Over-expression of cyclin E has been noted in several malignancies and is associated with high cell proliferation (Keyomarsi & Herliczek 1997, Nielsen et al. 1998). Therefore, this study used western blot analysis to determine that the increase in the G0/G1 cell population was inversely associated with the level of cyclin E-cdk2 proteins. Treatment of HepG2-TRα1#1, -TRα1#2 and -TRβ1 cells using 100 nM T3 led to a down-regulation of approximately 32 and 52% in the level of cdk2 protein following 24, and 48 h compared with T3-depleted media (Td) in HepG2-TRα1#1 cells (Fig. 2A, B). Similarly, 100 nM T3 led to down-regulation of approximately 27 and 31% in the protein level of cyclin E following 24 and 48 h (Fig. 2A, B). A similar effect was observed in the HepG2-TRβ1 (Fig. 2C), and HepG2-TRα1#2 cell lines (data not shown). Taken together, repression of both components of the cyclin E–cdk2 complex strongly agrees with the previous result of cell cycle blockage in G0/G1.
T3-repressed cell proliferation results from stimulation of p21 expression
Levels of the negative regulator of cell cycle progression, p21 protein, increase in senescent cells, while p21 over-expression has been demonstrated to block tumor cell growth (Gong et al. 2003). Thus, p21 was investigated as an additional target for controlling cell proliferation. p21 mRNA was strongly induced 1.3-, 3.9-and 7.3-fold in HepG2-TRα1#1 cells at 12, 24, and 48 h respectively, following the addition of 10 nM T3 to the media (Fig. 3A, B). Similarly, p21 protein was also significantly induced two- to five-fold by T3 treatment in two TRα1 stable cell lines (Fig. 3C, D). However, T3 did not markedly increase p21 mRNA or protein expression in the control cell line, HepG2-Neo (data not shown).
T3 treatment impacts the phosphorylation state of Rb protein
Rb tumor suppressor is a critical negative regulator of cellular proliferation. The Rb protein was de-phosphorylated (Fig. 4A) in HepG2-TRα1#1 or two other TR stable cell lines (data not shown) following T3 addition, possibly indicating that cyclin E was inactivated by p21. The expression of hyperphosphorylated Rb protein increased significantly following 24 or 48 h T3 treatment when HepG2-TRα1 cells were incubated in control conditions (Td) (Fig. 4A). As a further positive control, the phosphorylation status of Rb was investigated in the GC cell line. Consistent with previous studies (Barrera-Hernandez et al. 1999), Rb was hyperphosphorylated in GC cells after T3 treatment for 48 h (Fig. 4B). However, T3 did not significantly change the phosphorylation status of Rb in the control cell line, HepG2-Neo (data not shown). These experimental results indicate that the incubation of HepG2 cells over-expressing TR in media containing T3 represses the hyperphosphorylation of Rb.
TGF-β is stimulated by T3
To better understand how T3 inhibited the proliferation of HepG2-TRα1 cells, this study investigated the influence of T3 on the TGF-β expression. Similar to previously published results (Lin et al. 2004), TGF-β at the mRNA level was significantly stimulated about 1.5- to 3-fold by T3 in HepG2-TRα1#1 cells (Fig. 5A). Additionally, the 12 kDa TGF-β protein was up-regulated about two- to threefold 24 or 48 h following the addition of T3 (Fig. 5B). To further clarify the influence of T3 on TGF-β at the transcriptional level, the TGF-β 5′-flanking region (from −1362 to +101) was cloned into the pGL3 vector and its activity was assayed. T3 was demonstrated to increase the promoter activity by approximately 7.8- and 5.8-fold at 10 and 100 nM concentrations of T3 respectively in the TRα1 stable cell line, compared with its activity in control (Td) media without the addition of T3 (Fig. 5C). However, T3 did not considerably increase the promoter activity in the control cell line, HepG2-Neo (Fig. 5C).
TGF-β and its neutralizing antibody influence cyclin E, cdk2 and Rb expression
To clarify the signaling pathways involved in the repression of cyclin E, cdk2 and Rb by T3, this study investigated the involvement of TGF-β. The data indicate that treating cells with T3 for 48 h represses cdk2 expression by at least 40% at the protein level compared with the control (Td) conditions (Fig. 2A, B; Fig. 6A, B, C, lane 1 vs 2). Moreover, TGF-β alone also repressed the expression of cdk2 following 40 min treatment (Fig. 6A, B, C lane 1 vs 3). Notably, either T3 or TGF-β repressed the expression of cyclin E, cdk2, and rendered the Rb protein in the hypophosphorylated form (Fig. 6A, B, C lane 1 vs 2 and 3) in the HepG2-TR cells. Importantly, the repression of cyclin E, cdk2, and ppRb by T3 was blocked by the addition of TGF-β neutralizing antibody (nAb) (Fig. 6A, B, C lane 2 vs 6), but not by the non-specific antibody (nsAb) (Fig. 6A, B, C, lane 2 vs 5) in the HepG2-TRα1#1, -TRα1#2, and -TRβ1 stable lines. However, the effects of T3 and TGF-β were not observed in the Neo cells (Fig. 6D) and did not synergistically repress the expression of cyclin E, cdk2, and ppRb (Fig. 6A, B, C lane 4 vs 2 and 3) in the HepG2-TR cells. Moreover, TGF-β neutralizing antibody, but not the control antibody can reverse the cell growth inhibition effect of T3 (Fig. 1). Thus, cell proliferation is repressed by T3 through a TGF-β- mediated mechanism. Additionally, T3 controls the expression and activity of a number of cell cycle regulators via TGF-β, including p21, pRb and the cyclin E–cdk2 complex.
This study identified a novel pathway of T3 signaling mediated by TGF-β for inhibiting the proliferation of hepatoma cells expressing high levels of TR proteins. The effect of T3 treatment in promoting the proliferation of normal hepatocyes or GC cells derived from the pituitary has been well documented (Chou et al. 1987, Barrera-Hernandez et al. 1999). However, HepG2 and Hep3B cells do not express detectable TR proteins (Lin et al. 1994). Unlike previous studies, the results of this study indicate that T3 represses the proliferation of hepatoma cells rather than promoting it. The experimental data indicate that T3 only significantly suppresses the growth of HepG2-TR over-expressing cells. However, this T3-repressed effect was not observed in the control cell line (HepG2-Neo) that did not express detectable TR. The study does not contradict the results of in vivo studies reported by Ledda-Columbano et al.(2000). Their results demonstrated that T3 supplemented an increase in the BrdUrd-labeling index in the carcinogen-induced rat hepatocellular carcinoma (HCC). However, the TR expressing level in rat HCC is unknown (Ledda-Columbano et al. 2000). Actually, their data indicated that T3 administration, despite stimulating hepatocyte proliferation, resulted in a 70% reduction in the number of glutathione S-transferase (GST)-positive lesions, the marker enzyme used to identify pre-neoplastic lesion, with no increase in the size of the remaining nodules. In addition, repeated exposure of nodule-bearing rats to T3 caused a 50% reduction in the incidence of HCCs and 100% inhibition of lung metastasis. Their data also support the concept that T3-induced cell proliferation might not necessarily represent a promoting condition for putative pre-neoplastic lesions and demonstrates an anticarcinogenic effect of T3. To confirm that the suppressive effect of T3 treatments on hepatoma cell proliferation did not simply result from the toxic effects of this hormone, this study examined the expression of a number of factors that are known to be significantly involved in the cell cycle, for example cdk2, Rb, p21 and cyclin E. Additionally, this work used the GC cell line, which is known to proliferate when stimulated by T3 (Barrera-Hernandez et al. 1999). This study found that T3 repressed hepatoma cell growth by lengthening the G1 phase of the cell cycle, concomitantly decreasing the expression of cdk2 and cyclin E. To study the expression kinetics of the cell cycle, several regulatory factors were assayed, including proliferation cell nuclear antigen (PCNA), which represents an endogenous protein of the DNA polymerase δ. PCNA is an essential auxiliary protein of DNA polymerase δ, and is synthesized in the early G1 and S phases for processing of DNA replication. PCNA mRNA was repressed following T3 treatment in HepG2-TR stable lines (data not shown). This provides further support for the notion that T3 can suppress proliferation in cells that express TR.
cdk2 has been demonstrated to play a pivotal role in regulating cell cycle progression, is regulated by phosphorylation and can associate with cyclins A, E, D1, and D3. This investigation found that the inhibitory effect of T3 on cell proliferation occurs during the G0/G1 phase of the cell cycle. Interestingly, the cdk2–cyclinE complex is active in the G1 and S phases and is important for the progression from G1 to S (Harwell et al. 2004, Lents & Baldassare 2004). Additionally, cyclin E is pivotal in regulating the restriction point transition in the cell cycle. The role of cyclin E as a cdk2 activator in controlling restriction point transition in the cell cycle makes cyclin E an excellent candidate as a factor for involvement in tumor development. The hepatoma cell system presented here demonstrated reduced expression of both cyclin E and cdk2 following T3 application, although further study is required to determine whether this phenomenon is because of a direct or an indirect effect. Aberrant regulation of cyclin E is a common phenomenon seen in tumor cells and has been reported in tumor tissues isolated from breast cancer patients (Akli et al. 2004, Harwell et al. 2004). An interesting study by Pibiri et al.(2001) demonstrated that, in Wistar rats, hepatocyte proliferation induced by T3 occurred in the absence of AP-1, nuclear factor-κB, and STAT3 activation or any change in the mRNA levels of the immediate early genes c-fos, c-jun, and c-myc. However, this study found that T3 treatment increased cyclin D1 mRNA and protein levels, and moreover this increase occurred much more rapidly than liver regeneration following a two-thirds partial hepatectomy. Regrettably, the expression level of TGF-β was not examined. In contrast, in this investigation, we did not observe any significant change in the level of cyclin D1 expression following T3 treatment (data not shown). It is possible that T3 influences the late G1 but not the early G1 phase. The cyclin D1–cdk4, cdk-6 complexes are activated in early G1, whereas cyclin E–cdk2 is activated in late G1 (Sherr 1996, Martin-Castellanos & Moreno 1997). Therefore, our observations regarding hepatoma cells differ from those for normal regenerating rat liver cells. Summarizing the results of this and previous studies, T3 appears both to induce proliferation of normal hepatocytes and to suppress the proliferation of hepatoma cells with TR expression.
p21 can bind and inhibit each member of the cdk family. It also directly binds to PCNA and thus inhibits DNA replication. The present work showed that the expression of p21 was stimulated markedly by T3 at both the mRNA and protein levels, and may be, at least partially, responsible for blocking cell proliferation.
TGF-β is a pleiotropic cytokine that elicits a broad range of cellular responses, including cell growth, differentiation, and apoptosis. One of the biological effects of TGF-β is to inhibit epithelial cell proliferation by inducing cell cycle arrest (Sporn & Roberts 1992, Massague 1998). The effectors of TGF-β-induced cell growth inhibition are cyclin-dependent kinase inhibitors; among these inhibitors, p21Cip1 plays a major role in numerous biological contexts (Li et al. 1995). Additionally, Buzzard et al. (2003) demonstrated that T3 and other hormones induced the progressive accumulation of the cell cycle inhibitors p27Kip1 and p21Cip1 in Sertoli cells. However, the underlying mechanisms responsible for suppressing proliferation remain largely unknown (Gong et al. 2003). This study found that T3 acted on the TGF-β promoter to activate transcription. However, further work is required to determine whether this activation is due of a direct or an indirect effect. Therefore, in these hepatoma cells, the two signaling pathways are linked and TGF-β works downstream of the T3 pathway.
Rb is a nuclear phosphoprotein that undergoes differential phosphorylation during the cell cycle. Hypophosphorylated Rb protein complexes with E2F to inhibit its trans-activity on target genes and thus halts cell cycle progression. Barrera-Hernandez et al.(1999) reported that T3 increased the phosphorylation of Rb protein and thus stimulated GC cell line proliferation, similar to the results obtained here using these cells. By contrast, in this study T3 caused the accumulation of hypophosphorylated Rb protein, thus suppressing cell cycle progression in hepatoma cells overexpressing TR proteins.
Shimizu et al.(2004) reported that OSI-461 (a potent protein kinase G activator) enhanced the G0/G1 arrest resulting from acyclic retinoid (belonging to the thyroid/steroid super-family), and a combination of these agents synergistically decreased expression of cyclin D1 protein and mRNA, inhibited cyclin D1 promoter activity, reduced the level of hyperphosphorylated forms of the Rb protein and induced cellular levels of the p21Cip1 protein and mRNA in HepG2 cells. These observations mirror some of the results reported here.
Sumitani et al.(1994) reported that androgen significantly stimulates growth of the mouse mammary Shionogi carcinoma SC-3 cells. This androgen-induced growth is partially blocked by T3. TGF-β also inhibits SC-3 cell growth. Thus, they investigated whether T3 exerted its inhibitory effects on SC-3 cell growth through TGF-β mRNA expression. This study showed that T3 stimulated the expression of TGF-β at the mRNA and protein levels in HepG2-TR stable cells. Meanwhile, TGF-β1 exerted its inhibitory effects through down-regulation of PCNA, cdks, and cyclin E. Although this study did not demonstrate the effect of TGF-β neutralizing antibody on the growth of HepG2 cells, it did show that TGF-β antibody neutralizing T3 repressed cdk2.
In conclusion, this work provides evidence that T3 and its receptor mediates the suppression of hepatoma cell proliferation by TGF-β. The results presented here raise the possibility that T3, via its receptors and TGF-β, helps to regulate hepatocyte tumor growth and development.
The effect of T3 on cell cycle distribution in HepG2-TRα1, -TRβ1 and -Neo cells. Data are means ± s.e.
|Cell cycle distribution (%)|
|12 h||56.56 ± 1.33||29.56 ± 0.45|
|12 h + T3||66.77 ± 1.07||18.38 ± 0.51|
|24 h||61.35 ± 3.01||30.27 ± 2.73|
|24 h + T3||83.07 ± 1.51||8.87 ± 0.93|
|48 h||68.30 ± 1.41||25.06 ± 1.44|
|48 h + T3||91.91 ± 0.87||4.20 ± 0.48|
|12 h||63.79 ± 2.45||21.55 ± 0.52|
|12 h + T3||65.56 ± 2.53||19.93 ± 0.12|
|24 h||57.55 ± 4.37||30.49 ± 3.42|
|24 h + T3||60.74 ± 6.62||28.67 ± 3.29|
|48 h||74.30 ± 2.61||20.40 ± 1.66|
|48 h + T3||80.41 ± 4.55||15.19 ± 3.70|
|12 h||51.95 ± 5.43||33.07 ± 3.62|
|12 h + T3||62.40 ± 3.54||18.78 ± 3.58|
|24 h||69.13 ± 4.27||25.78 ± 6.10|
|24 h + T3||83.26 ± 3.82||10.90 ± 2.16|
|48 h||65.26 ± 4.91||24.90 ± 2.56|
|48 h + T3||84.66 ± 2.46||8.70 ± 1.46|
This work was supported by grants from Chang-Gung University, Taoyuan, Taiwan (CMRP 1332, NMRP 1074) and the National Science Council of the Republic of China (NSC 91–2320-B-182–041). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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