Hepatoma-derived growth factor/nucleolin axis as a novel oncogenic pathway in liver carcinogenesis.

Hepatoma-derived growth factor (HDGF) overexpression is involved in liver fibrosis and carcinogenesis. However, the receptor(s) and signaling for HDGF remain unclear. By using affinity chromatography and proteomic techniques, nucleolin (NCL) was identified and validated as a HDGF-interacting membrane protein in hepatoma cells. Exogenous HDGF elicited the membrane NCL accumulation within 0.5 hour by protein stabilization and transcriptional NCL upregulation within 24 hours. Blockade of surface NCL by antibodies neutralization potently suppressed HDGF uptake and HDGF-stimulated phosphatidylinositol 3-kinase (PI3K)/Akt signaling in hepatoma cells. By using rescectd hepatocellular carcinoma (HCC) tissues, immunohistochemical analysis revealed NCL overexpression was correlated with tumour grades, vascular invasion, serum alpha-fetoprotein levels and the poor survival in HCC patients. Multivariate analysis showed NCL was an independent prognostic factor for survival outcome of HCC patients after surgery. To delineate the role of NCL in liver carcinogenesis, ectopic NCL overexpression promoted the oncogenic behaviours and induced PI3K/Akt activation in hepatoma cells. Conversely, NCL knockdown by RNA interference attenuated the oncogenic behaviours and PI3K/Akt signaling, which could be partially rescued by exogenous HDGF supply. In summary, this study provides the first evidence that surface NCL transmits the oncogenic signaling of HDGF and facilitates a novel diagnostic and therapeutic target for HCC.

Involvement of phosphatase and tensin homolog deleted from chromosome 10 in rodent model of neuropathic pain.

BACKGROUND: Many cancer research studies have extensively examined the phosphatase and tensin homolog deleted from chromosome 10 (PTEN) pathway. There are only few reports that suggest that PTEN might affect pain; however, there is still a lack of evidence to show the role of PTEN for modulating pain. Here, we report a role for PTEN in a rodent model of neuropathic pain. RESULTS: We found that chronic constriction injury (CCI) surgery in rats could elicit downregulation of spinal PTEN as well as upregulation of phosphorylated PTEN (phospho-PTEN) and phosphorylated mammalian target of rapamycin (phospho-mTOR). After examining such changes in endogenous PTEN in neuropathic rats, we explored the effects of modulating the spinal PTEN pathway on nociceptive behaviors. The normal rats exhibited mechanical allodynia after intrathecal (i.t.) injection of adenovirus-mediated PTEN antisense oligonucleotide (Ad-antisense PTEN). These data indicate the importance of downregulation of spinal PTEN for nociception. Moreover, upregulation of spinal PTEN by i.t. adenovirus-mediated PTEN (Ad-PTEN) significantly prevented CCI-induced development of nociceptive sensitization, thermal hyperalgesia, mechanical allodynia, cold allodynia, and weight-bearing deficits in neuropathic rats. Furthermore, upregulation of spinal PTEN by i.t. Ad-PTEN significantly attenuated CCI-induced microglia and astrocyte activation, upregulation of tumor necrosis factor-α (TNF-α) and phospho-mTOR, and downregulation of PTEN in neuropathic rats 14 days post injury. CONCLUSIONS: These findings demonstrate that PTEN plays a key, beneficial role in a rodent model of neuropathic pain.

Downregulation of hepatoma-derived growth factor contributes to retarded lung metastasis via inhibition of epithelial-mesenchymal transition by systemic POMC gene delivery in melanoma.

The prognosis of malignant melanoma is poor due to high incidence of metastasis, underscoring the demand for development of novel therapeutic strategies. Stress hormone pro-opiomelanocortin (POMC) is the precursor for several anti-inflammatory peptides that hold promise for management of cancer-related diseases. The present study evaluated the antimetastatic potential and mechanism of POMC therapy for metastatic melanoma. Adenovirus-mediated POMC gene delivery potently inhibited the invasiveness of human and mouse melanoma cells. Moreover, after induction of lung metastasis, systemic POMC expression significantly reduced the foci formation and neovascularization in lungs. Mechanistic studies revealed that POMC therapy inhibited the epithelial-mesenchymal transition (EMT) of melanoma cells by upregulation of E-cadherin and downregulation of vimentin and α-smooth muscle actin (α-SMA). In addition, microarray analysis unveiled POMC gene transfer reduced the mRNA level of multiple prometastatic factors, including hepatoma-derived growth factor (HDGF). Cell culture and immunohistochemical studies further confirmed that POMC gene delivery significantly decreased the expression of HDGF in melanoma cells and tissues. Despite stimulating the invasion and EMT, exogenous HDGF supply only partially attenuated the POMC-mediated invasion inhibition and EMT change in melanoma cells. Finally, we delineated the contribution of melanocortins to POMC-induced inhibition of invasion, HDGF downregulation, and E-cadherin upregulation. Together, these results indicate that HDGF downregulation participates in POMC-induced suppression of metastasis and EMT in melanoma.

Up-regulation of hepatoma-derived growth factor facilitates tumor progression in malignant melanoma [corrected].

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression in developing melanoma remains unclear. In this study, human melanoma cell lines (A375, A2058, MEL-RM and MM200) showed higher levels of HDGF gene expression, whereas human epidermal melanocytes (HEMn) expressed less. Exogenous application of HDGF stimulated colony formation and invasion of human melanoma cells. Moreover, HDGF overexpression stimulated the degree of invasion and colony formation of B16-F10 melanoma cells whereas HDGF knockdown exerted opposite effects in vitro. To evaluate the effects of HDGF on tumour growth and metastasis in vivo, syngeneic mouse melanoma and metastatic melanoma models were performed by manipulating the gene expression of HDGF in melanoma cells. It was found that mice injected with HDGF-overexpressing melanoma cells had greater tumour growth and higher metastatic capability. In contrast, mice implanted with HDGF-depleted melanoma cells exhibited reduced tumor burden and lung metastasis. Histological analysis of excised tumors revealed higher degree of cell proliferation and neovascularization in HDGF-overexpressing melanoma. The present study provides evidence that HDGF promotes tumor progression of melanoma and targeting HDGF may constitute a novel strategy for the treatment of melanoma.

Hepatoma-derived growth factor stimulates podosome rosettes formation in NIH/3T3 cells through the activation of phosphatidylinositol 3-kinase/Akt pathway.

Hepatoma-derived growth factor (HDGF) stimulates the migration, invasion and metastasis in several types of cancer cells. However, the mechanism underlying HDGF-stimulated migration remains unclear. In this study, we investigated the influence of HDGF on cytoskeleton remodeling and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in non-transformed NIH/3T3 cells. Exogenous HDGF promoted the migration and the formation of dorsal ruffles and podosome rosettes. Besides, HDGF supply increased the PI3K expression and Akt phosphorylation in dose- and time-dependent manners. Application of LY294002, a PI3K inhibitor, attenuated the HDGF-induced migration, dorsal ruffles and podosome rosettes formation. Consistently, the HDGF-overexpressing NIH/3T3 transfectants exhibited significantly increased motility and elevated PI3K/Akt activities, which were repressed by LY294002 or adenovirus-mediated overexpression of endogenous PI3K antagonist, PTEN. In summary, HDGF elicits the activation of PI3K/Akt signaling cascade, thereby promoting cytoskeleton remodeling to stimulate cellular migration.

The expression and prognostic significance of hepatoma-derived growth factor in oral cancer.

Hepatoma-derived growth factor (HDGF) participates in oncogenic progression and represents a prognostic factor in several types of cancer. This study aimed to elucidate the role of HDGF during oral carcinogenesis. HDGF expression and the tumorigenic behaviors in human oral cell lines were investigated by immunoblotting, invasion and colony formation assays. Recombinant adenovirus vectors were employed to modulate the HDGF level in oral cancer cells. Immunohistochemical analysis using tissue microarray (TMA) consisting of surgically resected samples from 95 oral cancer patients was performed to delineate the correlation between HDGF expression and clinic-pathological parameters. HDGF expression was higher in malignant oral cancer cells than benign ones. Adenovirus-mediated HDGF overexpression and knockdown demonstrated the cellular HDGF level regulated the tumorigenic behaviors of oral cancer cells. Immunohistochemical analysis revealed increased HDGF expression in the nucleus and cytoplasm in oral cancer tissues. The nuclear HDGF expression was significantly correlated with tumor stage (P=0.004) and grade (P=0.013) while the cytoplasmic HDGF expression was associated with tumor necrosis (P=0.002). Kaplan-Meier analysis revealed that patients with high nuclear HDGF expression had significantly worse 5-year disease-specific survival (P=0.0069), metastasis-free survival (P=0.0168), and local recurrence-free survival (P=0.0047). Multivariate analysis indicated that the nuclear HDGF labeling index was an independent prognostic factor for disease-specific and local recurrence-free survival. HDGF overexpression contributes to the oncogenic processes in oral cancer cells and constitutes a novel prognostic factor for survival outcome of oral cancer patients.

Hepatoma-derived growth factor regulates breast cancer cell invasion by modulating epithelial--mesenchymal transition.

Hepatoma-derived growth factor (HDGF) participates in tumourigenesis but its role in breast cancer is unclear. We set out to elucidate the expression profile and function of HDGF during breast carcinogenesis. Immunoblot and immunohistochemical studies revealed elevated HDGF expression in human breast cancer cell lines and tissues. Nuclear HDGF labelling index was positively correlated with tumour grade, stage and proliferation index, but negatively correlated with survival rate in breast cancer patients. HDGF over-expression was associated with lymph node metastasis and represented an independent prognostic factor for tumour recurrence. Gene transfer studies were performed to elucidate the influence of cellular HDGF level on the malignant behaviour and epithelial-mesenchymal transition (EMT) of breast cancer cells. Adenovirus-mediated HDGF over-expression stimulated the invasiveness and colony formation of MCF-7 cells. Moreover, HDGF over-expression promoted breast cancer cell EMT by E-cadherin down-regulation and vimentin up-regulation. Conversely, HDGF knockdown by RNA interference in MDA-MB-231 cells attenuated the malignant behaviour and elicited EMT reversal by enhancing E-cadherin expression while depleting vimentin expression. Because HDGF is a secreted protein, we evaluated the cellular function of recombinant HDGF and found that exogenously supplied HDGF enhanced the invasiveness of breast cancer cells by down-regulating E-cadherin and up-regulating vimentin at transcriptional and translational levels. In contrast, blockade of HDGF secretion with an HDGF antibody inhibited the malignant behaviours and EMT. Finally, exogenous HDGF partially reversed benzyl isothiocyanate (BITC)-induced EMT suppression. HDGF over-expression may exert a prognostic role for tumour metastasis and recurrence in breast cancer by modulating EMT. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pro-opiomelanocortin gene delivery suppresses the growth of established Lewis lung carcinoma through a melanocortin-1 receptor-independent pathway.

BACKGROUND: Pro-opiomelanocortin (POMC) is the precursor of several neuropeptides, such as corticotropin, melanocyte-stimulating hormone and the endogenous opioid (ß-endorphin). Our previous studies have indicated that POMC gene delivery inhibited the progression and metastasis of B16-F10 melanoma via the α- melanocyte-stimulating hormone/melanortin-1 receptor (MC-1R) pathway. METHODS: In the present study, the therapeutic efficacy of POMC gene therapy was evaluated in mice bearing established Lewis lung carcinoma (LLC) models both in vitro and in vivo. We also investigated the MC-1R-independent mechanism underlying POMC gene therapy. RESULTS: We found that POMC gene delivery significantly inhibited the growth and colony formation in MC-1R-deficient LLC cells. In addition, POMC gene transfer effectively suppressed the growth of established LLC in mice. The inhibitory mechanisms underlying POMC gene delivery were attibuted to be inhibition of proliferation and the induction of apoptosis. Moreover, POMC gene delivery attenuated tumor ß-catenin signaling by reducing protein levels of ß-catenin and its downstream proto-oncogenes, including cyclin D1 and c-myc. Lastly, POMC gene delivery induced a significant suppression of tumor vasculature. CONCLUSIONS: These results support the existence of an MC-1R-independent pathway for POMC gene therapy, which further expands the therapeutic spectrum of POMC therapy for multiple types of cancer.

Gefitinib attenuates transforming growth factor-ß1-activated mitogen-activated protein kinases and mitogenesis in NRK-49F cells.

Transforming growth factor-ß (TGF-ß), TGF-ß receptor (TGF-ßR), and epidermal growth factor receptor (EGFR) are important in the pathogenesis of kidney fibrosis, a result of renal fibroblast activation. The EGFR kinase inhibitor gefitinib attenuates glomerular fibrosis in hypertensive rats whereas dominant-negative EGFR attenuates interstitial fibrosis in mouse with acute renal ischemia. Thus, we studied the effects and molecular mechanisms of gefitinib in TGF-ß1-induced mitogenesis and collagen production in normal rat kidney interstitial fibroblast (NRK-49F) cells. We found that TGF-ß1 increased cell mitogenesis. TGF-ß1 also time-dependently increased cyclin D1 protein expression. TGF-ß1 rapidly transactivated EGFR. SB431542 (a type I TGF-ßR kinase inhibitor) and SB203580 (a p38 kinase inhibitor) attenuated TGF-ß1-induced phosphorylation of Smad2/3 protein. SB431542 and gefitinib attenuated TGF-ß1-induced phosphorylation of ERK1/2 and p38 kinase. SB431542 and gefitinib also attenuated TGF-ß1-induced cyclin D1 protein expression. Moreover, SB431542, gefitinib, PD98059 (an ERK1/2 inhibitor), and SB203580 attenuated TGF-ß1-induced cell mitogenesis. Finally, SB431542 and gefitinib attenuated TGF-ß1-induced collagen production. We concluded that gefitinib attenuates TGF-ß1-induced cell mitogenesis via the EGFR-ERK1/2/p38 kinase pathway in NRK-49F cells. Moreover, gefitinib attenuates TGF-ß1-induced cyclin D1 protein expression and collagen production. Thus, gefitinib attenuates TGF-ß1-induced mitogenesis and collagen production in vitro.

Differential toll-like receptor 3 (TLR3) expression and apoptotic response to TLR3 agonist in human neuroblastoma cells.

BACKGROUND: Toll-like receptor-3 (TLR-3) is a critical component of innate immune system against dsRNA viruses and is expressed in the central nervous system. However, it remains unknown whether TLR3 may serve as a therapeutic target in human neuroblastoma (NB). METHODS: TLR3 expression in human NB samples was examined by immunohistochemical analysis. Quantitative RT-PCR and western blot was used to determine TLR3 expression in three human NB cell lines. The effect of TLR3 agonist, polyinosinic-polycytidylic acid (poly(I:C)), on the growth of human NB cells was evaluated by WST-1 cell proliferation assay, flow cytometry analysis, and immunoblot analysis. Blockade of TLR3 signaling was achieved using TLR3 neutralizing antibody, small interference RNA, and 2-aminopurine (2-AP), an inhibitor of protein kinase R (PKR), an interferon-induced, double-stranded RNA-activated protein kinase. RESULTS: In immunohistochemical studies, TLR3 mainly expressed in the cytoplasm of ganglion cells and in some neuroblastic cells, but not in the stromal cells in human NB tissues. Among three human NB cell lines analyzed, TLR3 was significantly up-regulated in SK-N-AS cells at mRNA and protein level compared with other two low TLR3- expressing NB cells. Treatment with poly(I:C) elicited significant growth inhibition and apoptosis only in high TLR3-expressing SK-N-AS cells, but not in low TLR3-expressing SK-N-FI and SK-N-DZ cells. Moreover, poly(I:C) treatment significantly stimulated the activities of PKR, interferon regulatory factor 3 (IRF-3) and caspase-3 in SK-N-AS cells. Application of TLR3 neutralizing antibody or small interference RNA (siRNA) reduced the poly(I:C)-induced inhibition of cell proliferation and apoptosis in SK-N-AS cells. On the contrary, ectopic TLR3 expression enhanced the sensitivity of low TLR3-expressing NB cells to poly(I:C). Finally, application of 2-AP attenuated the poly(I:C)-induced IRF-3 and caspase-3 activation in SK-N-AS cells. CONCLUSION: The present study demonstrates that TLR3 is expressed in a subset of NB cells. Besides, TLR3/PKR/IRF-3/capase-3 pathway is implicated in the selective cytotoxicity of TLR3 agonist towards high TLR3-expressing NB cells.

A label-free differential proteomic analysis of mouse bronchoalveolar lavage fluid exposed to ultrafine carbon black.

Ultrafine carbon black (ufCB) is a potential hazard to the lung. It causes changes in protein expression and it increases alveolar-capillary permeability in the lung. Label-free quantitative proteomic methods allow a sensitive and accurate analytical method for identifying and quantifying proteins in a protein mixture without chemically modifying the proteins. We used a label-free quantitative proteomic approach that combined and aligned LC-MS and LC-MS/MS spectra to analyze mouse bronchoalveolar lavage fluid (BALF) protein changes associated with exposure to ufCB. We developed a simple normalization method for quantification without spiking the internal standard. The intensities of unchanged peptides were used as normalization factors based on a statistical method to avoid the influence of peptides changed because of ufCB. LC-MS/MS spectra and then database searching were used to identify proteins. The relative abundances of the aligned peptides of identified proteins were determined using LC-MS spectra. We identified 132 proteins, of which 77 are reported for the first time. In addition, the expression of 15 inflammatory proteins and surfactant-associated proteins was regulated (i.e., 7 upregulated and 8 downregulated) compared with the controls. Several proteins not previously reported provide complementary information on the proteins present in mouse BALF, and they are potential biomarkers for the understanding of mechanisms involved in ufCB-induced lung disorders hypothesize that using the label-free quantitative proteomic approach introduced here is well suited for more rigorous, large-scale quantitative analysis of biological samples. We hypothesize that this label-free quantitative proteomic approach will be suited for a large-scale quantitative analysis of biological samples.

Advanced glycation end-products activate extracellular signal-regulated kinase via the oxidative stress-EGF receptor pathway in renal fibroblasts.

Advanced glycation end-products (AGEs), epidermal growth factor receptor (EGFR), reactive oxygen species (ROS), and extracellular signal-regulated kinases (ERK) are implicated in diabetic nephropathy (DN). Therefore, we asked if AGEs-induced ERK protein phosphorylation and mitogenesis are dependent on the receptor for AGEs (RAGE)-ROS-EGFR pathway in normal rat kidney interstitial fibroblast (NRK-49F) cells. We found that AGEs (100 microg/ml) activated EGFR and ERK1/2, which was attenuated by RAGE short-hairpin RNA (shRNA). AGEs also increased RAGE protein and intracellular ROS levels while RAGE shRNA and N-acetylcysteine (NAC) attenuated AGEs-induced intracellular ROS. Hydrogen peroxide (5-25 microM) increased RAGE protein level while activating both EGFR and ERK1/2. Low-dose hydrogen peroxide (5 microM) increased whereas high-dose hydrogen peroxide (100 microM) decreased mitogenesis at 3 days. AGEs-activated EGFR and ERK1/2 were attenuated by an anti-oxidant (NAC) and an EGFR inhibitor (Iressa). Moreover, AGEs-induced mitogenesis was attenuated by RAGE shRNA, NAC, Iressa, and an ERK1/2 inhibitor (PD98059). In conclusion, it was found that AGEs-induced mitogenesis is dependent on the RAGE-ROS-EGFR-ERK1/2 pathway whereas AGEs-activated ERK1/2 is dependent on the RAGE-ROS-EGFR pathway in NRK-49F cells.

Advanced glycation end-product-induced mitogenesis is dependent on Janus kinase 2-induced heat shock protein 70 in normal rat kidney interstitial fibroblast cells.

Kidney interstitial fibroblast proliferation is important in the pathogenesis of diabetic renal fibrosis. In this regard, advanced glycation end-product (AGE)-induced proliferation in normal rat kidney interstitial fibroblast (NRK-49F) cells is dependent on the Janus kinase 2 (JAK2) signal transducers and activators of transcription (STAT) pathway. Heat shock protein (Hsp) is a molecular target of JAK/STAT. Thus, the role of Hsp70 in AGE-induced mitogenesis in NRK-49F cells was studied. The AGE dose (100-200 microg/mL) and time (16-72 h) dependently increased Hsp70 protein expression. AGE-induced Hsp70 was attenuated by AG-490 (a JAK2 inhibitor) and N-acetylcysteine. AGE also increased tyrosine phosphorylation of Hsp70, cyclin E, and cyclin D1 (to a lesser extent) while increasing Hsp70 protein interactions with STAT1, STAT3, STAT5b, cyclin D1, and cyclin E. AGE-induced tyrosine phosphorylation of Hsp70 and cyclin E (but not cyclin D1) was attenuated by AG-490. AGE-induced mitogenesis, cyclin D1, and cyclin E were attenuated by Hsp70 antisense oligodeoxynucleotide and 2-aminopurine (an Hsp70 inhibitor). AGE-induced Hsp70 and mitogenesis were also attenuated by N-acetylcysteine. It was concluded that AGE-induced Hsp70 protein expression and tyrosine phosphorylation are dependent on JAK2 in NRK-49F cells. AGE increased protein-protein interactions among Hsp70, STAT1, STAT3, STAT5b, cyclin D1, and cyclin E. Moreover, AGE-induced mitogenesis is dependent on Hsp70 and oxidative stress.