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1 Department of Veterinary Medicine, College of Bio-Resources and Agriculture National Taiwan University, 10617 Taipei, Taiwan ROC
2 Department of Surgery, Mackay Memorial Hospital, 10449 Taipei, Taiwan ROC
3 Mackay Medicine, Nursing and Management College, 10449 Taipei, Taiwan ROC
4 Department of Surgery, National Taiwan University Hospital, National Taiwan University, 10002 Taipei, Taiwan ROC
(Requests for offprints should be addressed to I-C Guo; Email: iguo{at}ntu.edu.tw)
C Chen and Y-C Chang contributed equally to this work
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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-glutamyl transferase (Gonzalez et al. 2004). It appears that certain factors derived from adipose tissues plausibly play positive roles in liver tumorigenesis. Leptin, the major adipocyte-derived adipocytokine, is a known biomarker molecule of obesity due to the tight association of its circulating levels with body mass index and total body fat (van Gaal et al. 1999). At least, six transcript variants of leptin receptors (OBRs), which belong to class I cytokine receptor family, were identified and designed as OBRa-f (Fruhbeck 2006). Without an intrinsic tyrosine kinase activity, the liganded OBR usually activates a cytoplasmic kinase, Janus kinase 2 (JAK2), to transmit leptin signals intracellularly (Fruhbeck 2006). The expressions of OBRs variants had been detected in human normal hepatocytes (Briscoe et al. 2001), HCC cell lines (Cohen et al. 1996, Wang et al. 1997, Liu et al. 2004), as well as in human HCC tissues and their surrounding non-tumoral liver tissues (Wang et al. 2004). These studies indicated that both human non-malignant and malignant hepatocytes are capable of directly receiving leptin signaling. The responsiveness of human hepatocytes to physiological levels of leptin, primarily secreted by subcutaneous and abdominal adipose tissues, has been demonstrated (Chen et al. 2006b). Interestingly, leptin could be produced by HCC and normal liver tissues (Wang et al. 2004), as well as activated hepatic stellate cells (HSCs; Ding et al. 2005), possibly leading to the high accumulation of intrahepatic leptin; therefore, leptin signaling perhaps controls the growth of hepatocytes and HCC cells via endocrine, paracrine, and/or autocrine modes.
Actually, the growth factor-like functions of leptin had been observed in many types of cancer cells (Somasundar et al. 2004, Garofalo & Surmacz 2006). Given that JAK2-initiated signaling pathways, such as phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase 1/2 (ERK1/2) had been revealed to mediate proliferative and/or anti-apoptotic effects of leptin in various cancer cell types, such as breast (Chen et al. 2006a, Frankenberry et al. 2006), endometrium (Sharma et al. 2006), and neuroblastoma (Russo et al. 2004), we hypothesize that leptin might also have a somatotropic function in HCC cells possibly by activating JAK2-linked PI3K/Akt and MEK/ERK1/2 pathways. Although only one paper, to the best of our knowledge, had reported the proliferative effect of leptin on non-tumoral hepatic WRL-68 cells (Briscoe et al. 2001), no publication describes the somatotropic effect of leptin in HCC cells. In this paper, we are intended to verify the proliferative and anti-apoptotic effects of leptin and study the underlying mechanisms along with their signaling pathways in HCC cells. Here, we demonstrate that leptin directly stimulates proliferation and inhibits transforming growth factor (TGF)-ß 1-induced apoptosis of HCC cells. Our evidence show that leptin up-regulates cyclin D1 to speed up cell cycle progression and down-regulates Bax to reduce programed cell death. These leptin effects are plausibly mediated by JAK2-initiated signaling pathway that involves activations of PI3K/Akt and MEK/ERK1/2 in order of occurrence.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Human recombinant leptin, epidermal growth factor (EGF), tyrphostin AG490, U0126, wortmannin, LY294002, PD98059, monoclonal anti-ß-actin antibody (Sigma Chemical Co.), TGF-ß (R&D Systems Inc., Minneapolis, MN, USA), polyclonal antibodies against phospho-JAK2 (Upstate, Charlottesville, VA, USA), Bax, PARP, phospho-Akt, or phospho-ERK1/2 (Cell Signaling Technology, Beverly, MA, USA), JAK2 or ERK1/2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and monoclonal antibody against Akt, Bcl-2, or cyclin D1 (BD Pharmingen, Palo Alto, CA, USA) were commercially obtained.
Cell lines and culture
The human HCC cell lines, Hep3B and HepG2, as well as non-malignant hepatic cell line, Chang liver, were obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle medium (Invitrogen Co.) supplemented with 10% fetal calf serum (Biological Industries Ltd, Kibbutz Beit Haemek, Israel), 100 U/ml penicillin G, and 0.1 g/l streptomycin (Sigma Chemical Co.) in a 5% CO2 incubator at 37 ° C.
RT-PCR
Total cellular RNA was extracted with Trizol reagent (Invitrogen Co). The RT-PCR was performed with the published protocol (Chen et al. 2006a, Hsu et al. 2006). Briefly, 2 µg total RNA were denatured at 65 ° C for 20 min, immediately cooled on ice, and incubated with the reverse transcriptase reaction mixture (1 U RNase inhibitor, 1 mM dNTP, 1 µM poly-dT oligonucleotide; Roche Applied Science), and 0.4 U MMLV reverse transcriptase (Invitrogen Co.) in a total volume of 20 µl at 37° C for 90 min to generate the first-strand cDNA. Following stopped by 95 ° C for 5 min and rapidly cooled on ice, the reversely transcribed cDNA was further PCR-amplified by specific primers in 50 µl PCR mixture (cDNA, 0.25 mM dNTP, 1 µM forward and reverse primers, 0.5 U Taq). The paired primer sets included forward OBRb (5'-CCA GAA ACG TTT GAG CAT CT- 3 ' ) and reverse OBRb (5'-CAA AAG CAC ACC ACT CTC TC-3' ), or forward OBRa (5'-GAA GGA GTG GGA AAA CCA AAG-3' ) and reverse OBRa (5'-CCA CCAT ATG TTA ACT CTC AG-3' ).
MTT assay
After cells were seeded in 96-well plates, serum starved for 24 h, and then incubated with indicated reagent in serum-free media for 48 h, the cell viabilities were measured using the 3-(4,5-dimethylthiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) proliferation assay with the previous protocol (Chen et al. 2006a). In brief, after cells were collected and incubated in medium containing 2 mg/ml MTT reagent (Sigma Chemical Co.) at 37 ° C for 4 h, the formazan crystals converted from tetrazolium salts by viable cells were dissolved in dimethyl sulfoxide (150 µl/well) and their absorbance at 570 nm was measured by a microplate spectrophotometer to reflect cell viability.
Cell number counting
After seeded in six-well plates and treated with vehicle or 250 ng/ml recombinant human leptin, the cells were harvested at the indicated time points and stained with 0.5% Trypan blue (Biological Industries). Subsequently, the number of viable cells was counted under a light microscope (Chen et al. 2006a).
Flow cytometry
The previous protocol for flow cytometric analysis was followed (Chen et al. 2006a). Briefly, cells were starved in serum-free media for 1 day followed by treatment with indicated reagent. After 48 h, cells were trypsinized, washed twice with cold PBS, and fixed in 70% ethanol at – 20 ° C for permeabilization. After 18 h, cells were washed and resuspended in PBS containing RNase (0.01 mg/ml) and propidium iodide (50 µg/ml; Sigma Chemical Co.) at 37 ° C for 30 min. The cell cycle profile was determined by Becton Dickinson FACScan, and the data were analyzed with ModFit LT 3.1 (Becton Dickinson, St José, CA, USA).
Construction of pCD-1KLuc plasmid
The cyclin D1 promoter fragment from – 927 to + 39 bp was PCR amplified using restriction enzyme site-tailed-specific primers designed by ourselves according to the sequences deposited in the GenBank (Accession no. Z29078) and genomic DNA extracted from normal human leukocytes, and then inserted in front of the firefly luciferase reporter gene contained in a promoterless pGL3Basic vector (Promega Co.) to generate the pCD-1KLuc construct. The used primers were CD/SacI-927 (5'-GTC GAG CTC AGC TTT CCA TTC AGA-3' ) and CD/HindIII + 39 (5'-ACA AAG CTT CTG CTG CTC GCT GCT A-3' ).
Transfection and luciferase activity assay
After 1-day serum deprivation, plasmids of pCD-1KLuc or pGL3Basic, together with pRluc-C1 (Perkin–Elmer Life Sciences, Boston, MA, USA), a CMV promoter-driven renilla luciferase construct as an internal control, were co-transfected into Hep3B with Lipofectamine Plus (Invitrogen Co.) according to manufacturer’s instruction in 24-well plates, and then maintained in serum-free media. After 24 h, pGL3Basic-transfected cells (pGL3) were still maintained in serum-free media, but pCD-1KLuc-transfected cells were incubated with serum-free media containing indicated reagent. After 48-h treatment, the transfected cell extracts were harvested and subjected to the dual luminescence reporter gene assay system (Perkin–Elmer Life Sciences) with the manufacturer’s instruction for detecting their luciferase activities. The relative luciferase activities were calculated by normalizing firefly luciferase activities with renilla luciferase activities.
Western blotting analysis
The protocol for western blotting was modified from the previous publication (Guo et al. 2001). Briefly, cell lysates were extracted with the lysis buffer (10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM dithiothreitol, 0.2 mM phenyl-methylsulphonyl fluoride, 1 µg/ml aproptinin, 1 µg/ml leupeptin, 1 mM Na3VO4, and 1 mM NaF) and separated on 12% SDS–PAGE gel. After the electrophoresed proteins were electrotransferred onto the nitrocellulose membrane (Perkin–Elmer Life Sciences), the membrane was blocked with 5% skim milk in TBST (20 mM Tris–HCl (pH 7.6), 137 mM NaCl, and 0.1% Tween-20), probed with appropriate primary antibodies at 4 ° C overnight and bound with HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) at room temperature for 1 h. After reacted with a chemiluminescence substrate (Amersham Biosciences), the blot was autographed onto an X-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan).
Statistical analysis
Statistical analyses were calculated with Student’s t-test. All data are presented as the mean ± S.D. and statistically significant difference is indictated in figures and tables with the description in the figure legend.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The expressions of two major isoforms of leptin receptors, the long-form OBRb and the short-form OBRa, had been detected in cells of liver origin (Cohen et al. 1996, Wang et al. 1997, Briscoe et al. 2001, Liu et al. 2004). We confirmed mRNA expressions of OBRb and OBRa in human malignant HCC cell lines, Hep3B and HepG2, as well as non-malignant Chang liver cell line with RT-PCR (Fig. 1A
), suggesting that hepatic malignant and non-malignant cell lines are capable of receiving leptin signals, at least partly, via these two OBRs.
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Leptin functions as a growth factor in variety of cell types (Somasundar et al. 2004, Garofalo & Surmacz 2006), thus, we tested leptin effect on growth of Hep3B, HepG2, and Chang liver cells. The results from MTT assay and Trypan blue staining method, both of which were used to indirectly and directly respectively, determine the cell viability, and were expected to reflect the regulation of cell growth by leptin. The results of MTT assays showed a dose-dependent stimulation of cell growth by 48-h leptin treatment. The significant stimulation was observed from the dose as low as 31.25 ng/ml in Hep3B (Fig. 1B
) and HepG2 (Fig. 1C), but the higher dose up to 62.5 ng/ml in Chang liver (Fig. 1D). The leptin effect was doubly checked with Trypan blue staining for directly counting the viable cell number. The significant increment of cell number was observed over 3-day leptin treatment in Hep3B (Fig. 1E) and Chang liver (Fig. 1F). These results indicate that leptin exerts growth-stimulating effect on both hepatic malignant and non-malignant cells.
To understand whether leptin has any survival effect to sustain higher cell number in condition of serum starvation, Hep3B was pre-incubated (on day – 1) in serum-free medium for 1 day before treatment (from days – 1 to 0), and then treated with leptin or vehicle (control; on day 0) in serum-free medium for two days (from days 0 to 2). As shown in Fig. 1G, 2-day serum deprivation (from days – 1 to 1) did not cease the elevation of cell number in both leptin-treated and control groups; however, cell number of the leptin-treated group was significantly higher than that of the control group, presumably due to proliferative effect of leptin in majority. On the subsequent day 2, cell number of the control group fell down that implies cells are dying after 3-day serum deprivation; on the contrary, the leptin-treated group maintained cell number at the same level as that on day 1. Most likely, in addition to proliferative effect, the survival effect of leptin may also be involved in the maintenance of cell number over 3-day serum deprivation.
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). These results suggest that leptin promotes cell cycle progression to stimulate growth of HCC cells.
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Cyclin D1 is a growth sensor induced by growth factors and mitogens to accelerate G1 progression (Knudsen et al. 2006); therefore, we examined leptin effect on cyclin D1 expression. In Hep3B, the amounts of cyclin D1 protein, detected with western blotting analysis, were gradually increased by increasing doses of leptin (Fig. 2A). To understand if leptin controls cyclin D1 gene expression, we analyzed cyclin D1 promoter activity in response to leptin treatment. The luciferase activity of Hep3B transfected with pCD-1KLuc, a pGL3Basic backbone construct containing a reporter luciferase gene driven by 5'-flanking – 927/+ 39 region of cyclin D1 gene, in leptin-treated group was significantly higher than that in control group, and the comparable activity was observed in the group treated with EGF or 5% fetal calf serum, which supposedly contains a variety of growth factors (Fig. 2B). The parent vector pGL3Basic expressed background levels due to lack of promoter (Fig. 2B). These results suggest that leptin, like growth factors, up-regulates cyclin D1 gene expression, by which the hepatic cell cycle progression may be accelerated.
Leptin stimulates cell growth through activations of JAK2, PI3K/Akt, and MEK/ERK1/2
The JAK2/signal transducer and activator of transcription 3 (STAT3), PI3K/Akt, and MEK/ERK1/2 pathways mostly mediate intracellular leptin signaling in various cell types (Fruhbeck 2006); accordingly, we investigated their roles in the leptin-stimulated growth of hepatic cells. As measured with western blotting analyses, although the total protein amounts of JAK2, Akt, and ERK1/2 were quite refractory to leptin, their phosphorylated forms were dose-dependently induced by leptin in Hep3B (Fig. 3A) and Chang liver (Fig. 3B). Simultaneously, the time-course experiments showed that the levels of phosphorylated forms of JAK2, Akt, and ERK1/2 were increased as soon as 10 min in Hep3B (Fig. 3C) and 5 min in Chang liver (Fig. 3D) during leptin treatment. These results identify the activations of JAK2, Akt, and ERK1/2 signaling kinases by leptin in hepatic cells.
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), PI3K (wortmannin; Fig. 4B), or MEK1/2 (U0126; Fig. 4C). Similar results were seen in Chang liver (Fig. 4D–F). To avoid that the unspecific or unexpected effects of above-mentioned inhibitors misled the results of inhibition experiments, the inhibition experiments were also carried out with analogous pharmacological inhibitors. Similar to wortmannin and U0126, the PI3K inhibitor, LY294002 (Fig. 4G), and the MEK1/2 inhibitor, PD98059 (Fig. 4H) respectively, blocked the leptin-induced Hep3B cell growth. Taken together, these findings consistently reveal that leptin possibly activates the signaling pathways, including JAK2, PI3K/Akt, and MEK/ERK1/2 to stimulate growth of both hepatic malignant and non-malignant cells.
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The reports describing the linkage among JAK2, PI3K/Akt, and MEK/ERK1/2 pathways (Zhao et al. 2000, 2002, Menu et al. 2004, Sahu 2004, Pai et al. 2005) reminded us to inspect whether there is any connection among these signaling pathways to mediate leptin effect. With western blotting analyses, we observed that leptin effectively induced the levels of phosphorylated forms of JAK2, Akt, and ERK1/2 with little influence on their total protein amounts in both Hep3B (Fig. 5A, lanes 1 and 2) and Chang liver (Fig. 5B, lanes 1 and 2). As expected, pre-treatment with AG490, U0126, and wortmannin specifically disabled leptin for inducing phosphorylations of JAK2, ERK1/2, and Akt respectively (Fig. 5A and B, lanes 3–5). Furthermore, the JAK2-specific inhibitor AG490 concurrently prevented leptin-induced phosphorylations of JAK2, Akt, and ERK1/2 (Fig. 5A and B, lane 3), but the inhibitors specific for MEK/ERK1/2(U0126) and PI3K/Akt (wortmannin) did not affect leptin-induced phosphorylation of JAK2 (Fig. 5A and B, lanes 4 and 5). These results indicate that JAK2 plays as an upstream mediator of both PI3K/Akt and MEK/ERK1/2 in intrahepatocytic leptin signaling. In addition, U0126 prohibited leptin-induced phosphorylation of ERK1/2 without influence on leptin-induced phosphorylation of Akt (Fig. 5A and B, lane 4), but wortmannin concomitantly blocked leptin-induced phosphorylations of both Akt and ERK1/2 (Fig. 5A and B, lane 5). These observations suggest that PI3K/Akt locates upstream from MEK/ERK1/2 within leptin-activated signaling pathway. To ruling out the possibility that results of inhibition experiments were from non-specific effects of wortmannin and U0126, their corresponding inhibitors LY294002 and PD98059 were tested. Like U0126, pre-treatment of PD98059 completely prevented leptin-induced phosphorylation of ERK1/2 without reducing leptin induction on the amounts of phosphorylated forms of JAK2 and Akt (Fig. 5C, lane 3). On the other hand, pre-treatment of LY294002, just resembling wortmannin, entirely cancelled leptin induction on phosphorylations of both Akt and ERK1/2, but did not affect induction of phosphorylated JAK2 by leptin (Fig. 5C, lane 4). According to these constant findings, we propose that leptin probably triggers a JAK2-initiated signaling cascade, comprising PI3K/Akt and MEK/ERK1/2 in the order of crosstalking priority in hepatic cells.
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To clarify the role JAK2, PI3K/Akt, and MEK/ERK1/2 play in leptin-accelerated cell cycle progression, we examined the effects of their inhibitors on cyclin D1 expression and cell fate of Hep3B. All specific inhibitors of JAK2 (AG490), PI3K/Akt (wortmannin and LY294002), and MEK/ERK1/2 (U0126 and PD98059) blocked leptin induction on cyclin D1 protein amounts, detected with western blotting analyses (Fig. 6A and B). Similar inhibitions by AG490, wortmannin, and U0126 were observed on the cyclin D1 promoter activity, analyzed by reporter luciferase activity assays of pCD-1KLuc-transfected Hep3B (Fig. 6C). These results suggest that activations of JAK2, PI3K/Akt, and MEK/ERK1/2 perhaps mediate leptin induction on cyclin D1 gene expression of HCC cells.
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). Taken together, our findings suggest that the signaling pathways involving activations of JAK, PI3K/Akt, and MEK/ERK1/2 may mediate leptin stimulation to speed up cyclin D1-depenedent cell cycle progression in HCC cells.
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After verifying proliferative effect of leptin, we like to examine its anti-apoptotic effect. The MTT assays showed that cell viability of Hep3B was significantly decreased after treatment with TGF-ß 1 (Fig. 7A), an anti-proliferative and apoptogenic factor of many cell types including HCC (Lamboley et al. 2000, Zhang et al. 2006). The results from western blotting analyses showed that 116 kDa active poly(ADP-ribose) polymerase (PARP) was cleaved into two 89 and 28 kDa fragments by TGF-ß 1 treatment (Fig. 7B; the 28 kDa fragment not shown), indicating that TGF-ß 1 induces Hep3B apoptosis. The TGF-ß 1-induced apoptosis was accompanied with increased expression of Bax, a pro-apoptotic Bcl-2 family protein, in the analyses of western blotting (Fig. 7C). Since TGF-ß 1 did not affect expression of Bcl-2, an anti-apoptotic Bcl-2 family protein, TGF-ß 1 eventually reduced the ratio between anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) Bcl-2 family proteins (Fig. 7C). These results suggest that TGF-ß 1 induces Hep3B apoptosis, at least partly, through up-regulation of pro-apoptotic Bax and resulting reduction of the Bcl-2/Bax ratio. Interestingly, leptin rescued Hep3B from TGF-ß 1-induced cell death (Fig. 7A). Although proliferative effect of leptin may compensate TGF-ß 1-reduced cell viability to some extent, the reduction of TGF-ß 1-induced PARP cleavage by leptin (Fig. 7B) clearly indicates that anti-apoptotic effect of leptin, which down-regulated Bax expression and resulted in partial reverse of TGF-ß 1-reduced Bcl-2/Bax ratio (Fig. 7C), also contributes to maintain the number of cell population. Comparing with control (vehicle) group, treatment of leptin alone had little basal effects on expressions of Bax, Bcl-2 (Fig. 7D) and cleavage of PARP (Fig. 9A, lanes 1, 7, and 8).
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To know what signaling pathway leptin functions through to defend HCC cells against TGF-ß 1-induced apoptosis, we co-treated cells with specific inhibitors of leptin-signaling kinases. As detected by MTT assay, leptin significantly salvaged Hep3B from TGF-ß 1-induced cell death; moreover, all inhibitors, including AG490 (Fig. 8A), wortmannin (Fig. 8B), and U0126 (Fig. 8C), blocked the leptin rescue. Additionally, we found that leptin dose-dependently antagonized the TGF-ß 1 induction on PARP cleavage and Bax expression (Fig. 9A and B). Any one of AG490, wortmannin, and U0126 restored the leptin-suppressed TGF-ß 1 induction on PARP cleavage and Bax expression (Fig. 9A and B). All treatments had comparable Bcl-2 expression with that of control group (vehicle only; Fig. 9B). Apparently, the signaling pathway involving activations of JAK2, PI3K/Akt, and MEK/ERK1/2 mediates anti-apoptotic effect of leptin against TGF-ß 1-induced HCC apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Although it has been known that leptin functions like a growth factor in various types of cancer cells (Somasundar et al. 2004, Garofalo & Surmacz 2006), the cyclin D1-dependent proliferative effect of leptin is little reported. We verified the leptin stimulation on growth of hepatocytes and HCC cells (Fig. 1B–G), and confirmed the cyclin D1-dependent proliferative effect of leptin on HCC cells by showing that leptin induced cyclin D1 expression (Figs 2 and 6) and concomitantly shifted cell populations from G0/G1-phase to both S- and G2/M-phases (Table 1). The aberrant expression of cyclin D1, which functions as a sensor of growth factors and controls the cell cycle transition from G1- to S-phase, is a hallmark of carcinogenesis (Knudsen et al. 2006). Given that disruption of the regulatory system controlling G1-phase progression is a common event in human hepatocarcinogenesis and cyclin D1 overexpression plays a carcinogenic role in a subset (11–13%) of HCCs (Hui et al. 1998), our results that leptin stimulated HCC cells to highly express cyclin D1 well document the potential role of leptin in HCC development. Similar induction of cyclin D1 expression by leptin was observed in human breast cancer cells (Okumura et al. 2002, Chen et al. 2006a) and colon cancer HT-29 cell line (Rouet-Benzineb et al. 2004), as well as primary mouse HSCs (Saxena et al. 2004). In addition, our observations that the 5'-flanking – 927/+ 39 region of cyclin D1 promoter was commonly responsive to the stimulation by leptin, EGF, and serum in HCC cells (Fig. 2B) suggest that human cyclin D1 promoter activity is, at least partly, driven by interaction of its 5'-flanking 927 bp region, possibly containing multiple cis-elements or a common cis-element, with trans-acting factors activated by distinct growth factors.
TGF-ß 1 is an effective inducer for Hep3B apoptosis evaluated by several apoptotic characteristics like cleavage of PARP (Lamboley et al. 2000). In our experiments, TGF-ß 1 increased pro-apoptotic Bax expression without affecting Bcl-2 expression, leading to reduce the ratio between anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) Bcl-2 family proteins (Figs 7C and 9B). That reduced Bcl-2/Bax ratio facilitates the release of mitochondrial cytochrome C, which subsequently induces cascaded activations of caspases that cleave 116 kDa active PARP into two inactive 89 and 28 kDa fragments (Ferrer & Planas 2003, Harada & Grant 2003). The cleavage of PARP is one of the irreversible events resulting in programed cell death. Here, for the first time, we observe that leptin protects HCC cells from TGF-ß 1-induced apoptosis by down-regulating pro-apoptotic Bax expression and consequentially increasing the Bcl-2/Bax ratio (Figs 7 and 9). By the similar way, leptin also protects human osteoblastic cells (Gordeladze et al. 2002), rat enterocytes (Sukhotnik et al. 2006) and follicular cells (Almog et al. 2001), and bovine oocytes (Boelhauve et al. 2005) against apoptosis.
In comparison with the other five short isoforms of leptin receptors, the role of full-length OBRb in leptin signaling is well studied and recognized as the main signal mediator of leptin. Owing to lack of an intrinsic tyrosine kinase domain, the OBRb binds and activates cytoplasmic kinases, such as JAK2, which continuously initiates the subsequent signal pathways, primarily including STAT3, PI3K/Akt, and MEK/ERK1/2 (Fruhbeck 2006). These signal pathways are also commonly activated by the binding of tyrosine kinase receptors with distinct growth factors to promote proliferation of HCC cells through transactivation of cell cycle regulators like cyclin D1 (Roberts & Gores 2005). In the leptin-treated Hep3B and Chang liver, we find that leptin induced the phosphorylations of JAK2, Akt, and ERK1/2 (Fig. 3), and either inhibitor specific for JAK2, PI3K/Akt, or MEK/ERK1/2 completely blocked both leptin-induced cyclin D1 expression (Fig. 6) and cell proliferation (Fig. 4; Table 2). Obviously, the proliferative effect of leptin is mediated by activations of JAK2, PI3K/Akt, and MEK/ERK1/2; moreover, the pharmacological inhibition experiments reveal that JAK2 locates prior to PI3K/Akt and MEK/ERK1/2, as the JAK2 inhibitor AG490 also prevented phosphorylations of Akt and ERK1/2 from leptin activation (Fig. 5A and B, lane 3).
Activations of both JAK2-linked pathways, PI3K/Akt (Zhao et al. 2002, Sahu 2004, Hsu et al. 2006) and MEK/ERK1/2 (Husain et al. 2001, Pai et al. 2005), by leptin were frequently observed, but few reports showed their signaling crosstalks. A previous study observed that the specific MEK/ERK1/2 inhibitor blocked leptin-induced PI3K activity (Ktori et al. 2003). Dissimilarly, we do not see any effect of MEK/ERK1/2 inhibitors, U0126 and PD98059, on leptin-induced phosphorylation of Akt (Fig. 5A and B, lane 4; Fig. 5C, lane 3), but find that PI3K/Akt inhibitors, wortmannin and LY294002, blocked leptin-induced phosphorylation of ERK1/2 instead (Fig. 5A–C, lane 5; lane 4). As a result, in hepatocytes and HCC cells, PI3K/Akt plausibly plays an upstream mediator of MEK/ERK1/2 during leptin signaling. Taken together, we propose a signaling pathway, connecting JAK2, PI3K/Akt, and MEK/ERK1/2 with time priority, to mediate leptin stimulation on the cyclin D1-dependent proliferation of hepatocytes and HCC cells. Similar activations of JAK2, PI3K/Akt, and MEK/ERK1/2 were involved in leptin effect on proliferation of human cancer cells from breast (Chen et al. 2006a, Frankenberry et al. 2006) and endometrium (Sharma et al. 2006). Additionally, the specific inhibitors stop the behaviors of leptin against TGF-ß 1-induced apoptotic characters (Figs 8 and 9); therefore, we speculate that leptin carries out its anti-apoptotic effect as well as proliferative effect through the same JAK2–PI3K/Akt–MEK/ERK1/2 pathway in early signaling events. In good agreement with our results, leptin suppressed apoptosis of human neuroblastoma cells by down-regulating the apoptotic factors caspase-10 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) through activations of JAK2, PI3K/Akt, and MEK/ERK1/2 (Russo et al. 2004).
In certain cases, leptin effects might pass through complicated signaling crosstalks. Like human colon HT-29 cancer cells, the activations of JAK2, PI3K/Akt, and JNK were required for mediating both proliferative and anti-apoptotic effects of leptin; wherein activated JAK2 separately activated PI3K/Akt and STAT3 but not c-Jun N-terminal kinase (JNK), and JNK increased activating protein-1 (AP-1) transcriptional activity when activated (Ogunwobi & Beales 2007). In that case, the involved signaling pathways, such as JAK2-STAT3, JAK2-PI3K/Akt, and JNK-AP-1, function as paralleled linkages instead of series connection. Although we do not forget about the possible roles of the other pathways in leptin effects and cannot rule out the possibility that pleiotropic effects of leptin could be a consequence of the entangled relationships among leptin signaling pathways, the essential role of the JAK2–PI3K/Akt–MEK/ERK1/2 linkage in the early signaling events of leptin effects was certainly verified in the studied HCC cells. This convinced hierarchy of leptin signaling pathway in fact provides sufficient advantage for making focus strategies to fight obesity-related or/and cirrhosis-associated liver cancer.
Contrary to our results, the anti-tumor effect of leptin on HCC in vivo and in vitro has currently been reported. Compared with the control groups, the leptin-treated groups of Hep3B-transplanted mice had reduced the tumor size, improved the survival rate, and significantly increased the peripheral natural killer cells (Elinav et al. 2006). We address the possibility whether the reported anti-tumor effect actually resulted from the natural killer cell-mediated immune defense of mouse host to reject human Hep3B xenograft, rather than an inhibitory effect of leptin on hepatocellular carcinogenesis. However, an in vitro inhibition of Hep3B growth by leptin was simultaneously displayed in that paper. The reason causing distinct results from ours is unclear. Nevertheless, we have identified the proliferative effect of leptin in two HCC (HepG2 and Hep3B) and one non-malignant Chang liver cell lines, as well as anti-apoptotic effect of leptin in Hep3B. Our results from pharmacological inhibition experiments consistently demonstrate that leptin promotes hepatocytic cell cycle progression by up-regulation of cyclin D1 and reduces programed cell death by down-regulation of Bax through a JAK2–PI3K/Akt–MEK/ERK1/2 cascaded pathway (Fig. 10). Combining both proliferative and anti-apoptotic effects, it is convincing that leptin potentially functions as a growth factor of HCC cells. Further studies are expected to understand the reasons for the conflicting observations and the reality of leptin function on liver tumorigenesis.
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Acknowledgements |
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