Downregulation of hepatoma-derived growth factor activates the Bad-mediated apoptotic pathway in human cancer cells
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Hepatoma-derived growth factor (HDGF) is highly expressed in human cancer and its expression is correlated with poor prognosis of cancer. The growth factor is known to stimulate cell growth while the underlying mechanism is however not clear. Transfection with HDGF cDNA stimulated while its specific antisense oligonucleotides repressed the growth of human hepatocellular carcinoma HepG2 cells. Furthermore, knock-down of HDGF by antisense oligos also induced apoptosis in HepG2 cells and in other human cancer cells, e.g. human squamous carcinoma A431 cells. HDGF knock-down was found to induce the expression of the pro-apoptotic protein Bad and also inactivate ERK and Akt, which in turn led to dephosphorylation of Bad at Ser-112, Ser-136, and activation of the intrinsic apoptotic pathway, i.e. depolarization of the mitochondrial membrane, release of mitochondrial cytochrome c, increase in the processing of caspase 9 and 3. As HDGF knock-down not only suppresses the growth but also induces apoptosis in human cancer cells, HDGF may therefore serve as a survival factor for human cancer cells and a potential target for cancer therapy.
KeywordsHDGF Apoptosis Bad
Hepatoma-derived growth factor (HDGF) is an acidic heparin-binding growth factor originally isolated from the conditioned media of the human hepatoma cell line, HuH-7 . HDGF belongs to the HDGF-related protein family. Members of this family included human HDGF, murine HRP-1, murine HRP-2, murine HRP-3, bovine HRP-4 and lens epithelium-derived growth factor (LEDGF), which was also reported to be highly expressed in human cancer cells. These proteins share a common N-terminal homologous to the HDGF amino terminus (HATH) domain of ~100 amino acid residues with a high level of sequence identity (66–89%), but the C-terminal regions show considerable variation in length and charge .
HDGF is expressed in a broad range of tissues, with the highest expression in the liver, lung, kidney, testis and placenta . The growth factor was reported to have mitogenic activity. In addition to hepatoma cells , recombinant HDGF was able to stimulate the growth of fibroblasts , vascular smooth muscle cells  and endothelial cells . HDGF was involved in the regulation of liver development by stimulating the proliferation of fetal hepatocyte . Moreover, the growth factor also facilitated the release of pro-apoptotic factors from mitochondria in TNF treated cells . It was reported that HDGF acts primarily in the nucleus and the translocation of the growth factor into the nucleus is important for its growth-promoting function [9, 10]. This is supported as the 240 amino acid HDGF protein contains a bipartite nuclear localization sequence and a DNA-binding PWWP domain .
Over-expression of HDGF can be detected in a number of human cancers including hepatoma [12, 13, 14, 15], gastric cancer , colorectal cancer , lung cancer  and melanoma . Increased expression of HDGF was found to be correlated with high proliferating states of the cancer cells and poor clinical outcome. Therefore, the expression of HDGF was reported to be a potential prognostic factor for hepatoma [12, 13, 14], non-small-cell lung cancer , esophageal cancer , gastric cancer  and pancreatic cancer .
HDGF was believed to be involved in the development of cancer. A gradual increase in HDGF expression was detected before the onset of liver tumor development in FLS mice . Over-expression of HDGF in 3T3 fibroblast cells conferred a transformed phenotype and enabled the formation of tumors in nude mice . Moreover, the HDGF level in the poorly differentiated human liver cancer is less as compared with that in the well-differentiated subtype . HDGF was reported to stimulate the growth of vascular endothelial cells  and elevated expression of the growth factor may therefore stimulate the angiogenic activity and promote the aggression of tumor. In addition, knock-down of HDGF was reported to suppress the proliferation of hepatoma cells  and inhibit the anchorage-independent growth of human lung cancer cells . Therefore, HDGF is suggested to play an important role in cancer development.
Although HDGF is over-expressed in human cancer and its over-expression is correlated with the development, progression and poor prognosis of human cancer, the mechanism of how HDGF may affect the growth of human cancer cells is still not clear. In the present study, the role of HDGF in the regulation of cancer cell proliferation and its underlying mechanism were investigated. It was found that knock-down of HDGF by HDGF antisense oligos not only inhibited the growth but also induced apoptosis in human cancer cells through the Bad mediated intrinsic apoptotic pathway. The results suggest that HDGF may function as a survival factor for human cancer cells and thus a potential target for cancer therapy.
Materials and methods
Human hepatocellular carcinoma HepG2 cells, RHepG2 cells and Hep3B were cultured in Dulbecco’s modified essential medium (DMEM) supplemented with 5% fetal bovine serum (Invitrogen, USA) and L-glutamine (Invitrogen, USA) at 37°C in a humidified atmosphere with 10% CO2. RHepG2 cells were the drug resistant subline of HepG2 cells. Human squamous cell carcinoma A431 cells, cervical carcinoma CaSki cells, SiHa cells, and lung carcinoma Calu-6 cells were cultured in the same condition except that 10% fetal bovine serum was used.
HDGF antisense oligonucleotides transfection
The sequence for the 21-base HDGF antisense oligos is complementary to the coding region of hdgf (nt 1027-1038, 5′-GGTTGGATCGCGACATGGCGG -3′) while the sequence of the sense oligos is the same as the coding sequence of hdgf (5′-CCGCCATGTCGCGATCCAACC-3′). All bases were phosphorothiolated to increase the stability of the nucleotides (TechDragon, Hong Kong). About 2 × 105 cells were seeded in 60 mm Petri dish for 48 h before the transfection. The cells were transfected with 0.2 μM HDGF antisense or sense oligos with the aid of oligofectamine (Invitrogen, USA) for 24 h at 37°C in serum-free DMEM.
HDGF full length cDNA transfection
The full-length HDGF cDNA in pcDNA3 was purchased from ATCC. The cells were seeded for 48 h before the transfection. The cells were transfected with 0.1 μg HDGF cDNA carrying vector (HDGF-pcDNA3) or empty vector (pcDNA3) with the aid of lipofectamine (Invitrogen, USA) for 24 h at 37°C in DMEM with 5% fetal bovine serum.
MTT cell viability assay
After transfection, the cells were allowed to grow for 3 days. After that, the cells were treated with 50 µl of 0.1 mg/ml 3-(4,5-Dimethylthiazole-2-yle)-2,5-diphenyltetrazolium bromide solution (USB, USA) at 37°C for 3 h and then lysed in 150 µl of dimethyl sulfoxide at room temperature for 30 min. The absorbance of each well was measured at 570 nm in a microplate reader.
DNA fragmentation assay
The cells were collected by trypsinization and lysed by lysis solution with 5 mM Tris–HCl buffer (pH 8.0), 100 mM EDTA (pH 8.0), 1% SDS and 0.2 mg/ml proteinase K. After incubation at 45°C for 2 h, the DNA in the crude lysate was extracted by phenol-chloroform and then precipitated by ethanol. The DNA extracted was dissolved in Tris–EDTA buffer with 0.2 mg/ml RNaseA and was incubated in 37°C overnight. Thereafter, 30 µg DNA was loaded into 1.5% EtBr staining agarose gel for electrophoresis. The DNA fragmentation pattern was viewed under UV light box.
Annexin V binding assay
The cells were collected by trypsinization. After washing with PBS, the cells were resuspended in Annexin V binding buffer containing 10 mM Hepes, 140 mM NaCl and 2.5 mM CaCl2 and stained with 5 µl Annexin V-GFP and 1 µg/ml propidium iodide at room temperature for 15 min. The cells were subjected to flow cytometry using the BD FACSCanto flow cytometer (Becton Dickinson, USA).
The cells were lysed by Tri Reagent (Molecular Research Centre, USA) for RNA extraction. After that, total RNA was subjected to reverse transcription with oligo dT primer (Invitrogen, USA) by MMLV Reverse Transcriptase (Promega, USA) at 42°C for 1 h and heated to 70°C for 15 min. PCR amplification of cDNAs using the pair of primers specific to hdgf within its open reading frame was carried out for 25 cycles of 94°C for 30 s, 55°C for 30 s and 70°C for 1 min; forward primer (5′-CCGCCATGTCGCGATCCAACC-3′) and reverse primer (5′-GGTTGGATCGCGACATGGCGG-3′). RT-PCR assay with primers specific for β-actin was also performed.
JC-1 staining was used to determine the mitochondrial membrane potential of cells. The cells were collected by trypsinization, resuspended in PBS, and stained with 10 µM JC-1 dye at 37°C for 15 min. Thereafter, the cells were subjected to flow cytometric analysis using the BD FACSCanto flow cytometer (Becton Dickinson, USA).
Mitochondrial cytochrome c release measurement
The cells were collected by trypsinization and were partially lysed by using 0.5 mg/ml digitonin (Sigma, USA) together with lysis solution containing 30 mM NaCl, 0.4 mM NaH2PO4, 3 mM Na2HPO4, 0.1 M sucrose, 2 µg/ml leupeptin, 8 µg/ml aprotinin and 0.4 mM phenylmethylsulphonyl fluoride (PMSF). The cell lysate was centrifuged and the level of cytochrome c was measured by Western blot analysis.
Western blot analysis
The cells were lysed in lysis solution containing 1% Triton X-100, 4.9 mM MgCl2, 1 mM sodium ortho-vanadate, and protease inhibitors aproptinin (21 µg/ml), leupeptin (0.5 µg/ml), and 1 mM PMSF. The cell lysate was centrifuged and the protein content in the supernatant was determined by the BCA Pierce Assay Kit (Pierce, USA). Protein from each sample was separated by SDS-PAGE according to size and was transferred onto PVDF membrane (Millipore, USA) by electro-blotting. The membrane was incubated with 5% non-fat dry milk in PBS at 4°C overnight and then probed with primary antibody for 2 h and with respective horse-radish peroxidase conjugated secondary antibody for 1 h. The primary antibodies are anti-cytochrome c, caspase 3, Bad, Bax, Bcl-2, Bcl-xL, ERK and pERK (Santa Cruz, USA), caspase 9 (stressgene, USA), pBad S112, pBad S136, pBad S155, Akt and pAkt S473 (Cell signaling, USA) and β-actin (Sigma, USA). The protein bands were detected by the ECL Western blot detection reagent (Amersham pharmacia, USA). The protein band intensity was scanned and analyzed by ImageJ (version 1.38×) software.
HDGF regulated the growth of HepG2 cells
Knock-down of HDGF induced apoptosis in human cancer cells
Knock-down of HDGF on the expressions of Bcl-2 family proteins
Knock-down of HDGF activated the intrinsic apoptotic pathway
Knock-down of HDGF regulated the phosphorylation status of Bad
EGF on phosphorylation of Bad upon HDGF knock-down
EGF on phosphorylation of ERK and Akt upon HDGF knock-down
HDGF may act as a survival factor for human cancer cells by regulating the process of apoptosis. Knock-down of HDGF was found to suppress the growth (Fig. 1) and trigger apoptotic cell death in human cancer cells (Figs. 2 and 3). The results for cell growth reduction by HDGF knock-down was in agreement with the work of Kishima . Besides this, knock-down of HDGF also reduced the anchorage independent growth of HepG2 cells (data not shown) as that been reported in the lung cancer cells . Although apoptosis induction might be one of the many ways that lead to the growth suppression in cells, the present study is the first to demonstrate that knock-down of HDGF will induce apoptosis in human cancer cells. Similar report was found in the knock-down of midkine (MK), a heparin binding growth factor, which is also highly expressed in a broad range of cancer cells. Knock-down of midkine induced apoptosis in gastric cancer cell lines . Since knock-down of HDGF not only suppressed the growth but also induced apoptosis in cancer cells, HDGF is therefore believed to function as a survival factor for human cancer cells.
The apoptosis induced by knock-down of HDGF is mediated by Bad, a pro-apoptotic Bcl-2 family protein, through the activation of the intrinsic apoptotic pathway. Knock-down of HDGF induced the expression (Fig. 4) and the dephosphorylation of Bad (Fig. 6). Bad is known to trigger apoptotsis by dimerizing with Bcl-2 and Bcl-xL as to neutralize their anti-apoptotic effects and thus promotes the pore formation on the mitochondria membrane, causes mitochondria membrane depolarization, and eventually activated the intrinsic apoptotic pathway [33, 34]. As expected, the apoptosis induced by knock-down of HDGF is associated with the activation of the intrinsic apoptotic pathway as knock-down of HDGF triggered the depolarization of the mitochondrial membrane (Fig. 5a), release of mitochondrial cytochrome c (Fig. 5b) and the processing of caspases 9 and 3 (Fig. 5c, d), which are hallmarks of the intrinsic apoptotic pathway.
Bad dephosphorylation together with Bad upregulation are critical for HDGF effect on apoptosis. The activity of Bad was reported to be regulated by phosphorylation at three serine residues, Ser-112, Ser-136 and Ser-155 . Phosphorylation of Bad at Ser-112 and Ser-136 stimulates the binding of Bad with 14-3-3 proteins and sequesters Bad in the cytosol while the phosphorylation of Bad at Ser-155 inhibits the direct interaction of Bad with Bcl-2 and Bcl-xL . Phosphorylation of Bad at any these three serine residues prevents Bad from triggering apoptosis. On the contrary, dephosphorylation of Bad leads to the release of 14-3-3 protein and allows Bad to gather at the mitochondria where it dimerizes with Bcl-2 and Bcl-xL to trigger the depolarization of the mitochondrial membrane which eventually activates the intrinsic apoptotic pathway [35, 36]. Dephosphorylation of Bad at Ser-112 and Ser-136 rather than Ser-155 was found to be important in mediating apoptosis induced by HDGF knock-down. The levels of Ser-112 and Ser-136 dephosphoryation on Bad upon HDGF knock-down are higher that that at Ser-155. EGF stimulated the phosphorylation of Bad at Ser-112 and Ser-136 but not Ser-155  while forskolin stimulated the phosphorylation of Bad at Ser-155 but not at Ser-112 and Ser-136 (Fig. 7). EGF partially suppressed the apoptosis induced by HDGF knock-down while forskolin cannot (Fig. 8). All these indicate that dephosphorylation of Bad at Ser-112 and Ser-136 rather than Ser-155 is important in mediating apoptosis induced by HDGF knock-down. The significance of Bad upregulation in HDGF knock-down induced apoptosis is hinted as EGF only partially overcomes the HDGF knock-down effect on apoptosis and has no effect on Bad upregulation. The role of Bad is further confirmed as the level of apoptosis induced by HDGF knock-down is partially reduced by co-silencing of Bad (Supplementary Fig. 2).
Knock-down of HDGF, through inactivation of the Ras/Raf/MEK/ERK and PI3K/Akt pathways, induced dephosphorylation of Bad at Ser-112 and Ser-136. According to the literature, phosphorylation of Bad at different serine residues was regulated by different signaling pathways. Phosphorylation of Bad at Ser-112, Ser-136 was at least regulated by the Ras/Raf/MEK/ERK and PI3K/Akt pathways respectively [28, 29]. In addition to Bad phosphorylation, knock-down of HDGF was found to reduce the phosphorylation of ERK and Akt. EGF induced the ERK and Akt phosphorylation and at the same time induced the phosphorylation of Bad at Ser-112 and Ser-136 in HepG2 cells (Fig. 7). Besides this, EGF was found to reverse the decrease in phosphorylation of ERK and Akt upon HDGF knock-down (Fig. 10b). Since EGF but not forskolin can suppress the level of apoptosis induced by HDGF knock-down (Fig. 8), it further supports that inactivation of the Ras/Raf/MEK/ERK and PI3K/Akt may be upstream to Bad dephosphorylation at Ser-112 and Ser-136 that eventually leads to activation of the intrinsic apoptotic pathway upon HDGF knock-down. On the other hand, the role of protein phosphatases that are known to induce dephosphorylation of Bad is not yet defined.
The results of our study indicate that HDGF is important for the growth of human cancer cells probably through the regulation of the intracellular signaling pathways. However, the mechanism of how HDGF regulates the signaling pathways remains to be further investigated. It was suggested that HDGF may act in an autocrine or paracrine manner that it may be secreted out of the cells and bind on the cell surface receptor to activate the intracellular signaling pathways . However, the receptor of HDGF has not yet been identified and this hypothetic mechanism is still needed to be further confirmed. Besides this, knock-down of HDGF may alter the expression of genes that regulate the intracellular signaling pathway. According to Zhang’s study in lung cancer cells , knock-down of HDGF was associated with the down-regulation of AXL, a receptor tyrosine kinase, and thus affects the intracellular signaling pathway. On the other hand, HDGF may regulate the gene transcription directly as previously HDGF was detected in the nucleus of cells and the ability of HDGF translocation into the nucleus is important for its growth promoting activity . It was also reported that HDGF may regulate gene expression directly by binding with the promoter region of the target genes .
Although the mechanism for HDGF to regulate the intracellular signaling pathways is yet to be defined, our study however indicates that HDGF may act as a survival factor for human cancer cells. This may account for the poor prognosis and low survival rate for those cancer patients with HDGF over-expression. Furthermore, the study also indicates the possible use of HDGF as the potential target in cancer therapy.
This work was supported by a Grant from the Shanghai-Hong Kong Anson Research Foundation, Hong Kong.
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