Inhibition Of Hepatocyte Growth Factor Research Articles

CCL20 induces colorectal cancer neoplastic epithelial cell proliferation, migration, and further CCL20 production through autocrine HGF-c-Met and MSP-MSPR signaling pathways.

CCL20-CCR6 interactions promote colorectal cancer through direct effects on neoplastic epithelial cells and through modulating the tumor microenvironment. The mechanism of these effects on neoplastic epithelial cells is poorly understood. This study demonstrates that CCL20 induces secretion of hepatocyte growth factor (HGF) and phosphorylation of HGF’s cognate receptor c-Met in HT29 and HCT116 colorectal cancer cell lines both in concentration- and time-dependent manners. Similar to CCL20, HGF induces migration, autofeedback CCL20 secretion, and ERK1/2 phosphorylation in the colon cancer cells. CCL20-dependent ERK1/2 phosphorylation is blocked by HGF inhibition, and CCL20-dependent migration and CCL20 secretion are blocked by inhibition of HGF or ERK. Interestingly, unlike CCL20, HGF does not induce proliferation of colon cancer cells, and CCL20-dependent cell proliferation is not blocked by direct HGF inhibition. CCL20-dependent proliferation, however, is blocked by the multi-tyrosine kinase inhibitor crizotinib. Exploring this effect, it was found that CCL20 also induces production of MSP and phosphorylation of MSP’s receptor MSPR by the colorectal cancer cells. CCL20-dependent cell proliferation is inhibited by directly blocking MSP-MSPR interactions. Thus, CCL20-mediated migration and CCL20 secretion are regulated through a pathway involving HGF, c-Met, and ERK, while CCL20-mediated proliferation is instead regulated through MSP and its receptor MSPR.

Knockout of Hepatocyte Growth Factor by CRISPR/Cas9 System Induces Apoptosis in Hepatocellular Carcinoma Cells.

Background: CRISPR/Cas9 system is a prokaryotic adaptive immune response system that uses noncoding RNAs to guide the Cas9 nuclease to induce site-specific DNA cleavage. Hepatocyte growth factor (HGF) is a well-known growth factor that plays a crucial role in cell growth and organ development. According to recent studies, it has been reported that HGF promoted growth of hepatocellular carcinoma (HCC) cells. Here, we investigated the apoptotic effects in HCC cells. Methods: Crispr-HGF plasmid was constructed using GeneArt CRISPR Nuclease Vector. pMex-HGF plasmid that targets HGF overexpressing gene were designed with pMex-neo plasmid. We performed real time-polymerase chain reaction to measure the expression of HGF mRNA. We performed cell counting assay and colony formation assay to evaluate cell proliferation. We also carried out migration assay and invasion assay to reveal the inhibitory effects of Crispr-HGF in HCC cells. Furthermore, we performed cell cycle analysis to detect transfection of Crispr-HGF induced cell cycle arrest. Collectively, we performed annexin V/PI staining assay and Western blot assay. Results: In Crispr-HGF-transfected group, the mRNA expression levels of HGF were markedly downregulated compared to pMex-HGF-transfected group. Moreover, Crispr-HGF inhibited cell viability in HCC cells. We detected that wound area and invaded cells were suppressed in Crispr-HGF-transfected cells. The results showed that transfection of Crispr-HGF induced cell cycle arrest and apoptosis in HCC cells. Expression of the phosphorylation of mitogen activated protein kinases and c-Met protein was regulated in Crispr-HGF-transfected group. Interestingly, we found that the expression of HGF protein in conditioned media significantly decreased in Crispr-HGF-transfected group. Conclusions: Taken together, we found that inhibition of HGF through transfection of Crispr-HGF suppressed cell proliferation and induced apoptotic effects in HCC Huh7 and Hep3B cells.

HGF/c-Met Inhibition as Adjuvant Therapy Improves Outcomes in an Orthotopic Mouse Model of Pancreatic Cancer.

Simple SummaryPancreatic cancer (PC) has a poor prognosis. Even though surgical resection and adjuvant chemotherapy is the most effective therapy, recurrence remains common. In this paper, we investigate the effectiveness of dual inhibition of hepatocyte growth factor (HGF) and c-MET when used as treatment after surgical resection of PC in mice. The HGF/c-Met pathway is a major mediator of pancreatic stellate cell (stromal cell)—PC cell interactions. Using single-cell RNA sequencing, we also investigated the existence of co-metastasising cells, circulating pancreatic stellate cells (cPSCs), as facilitators of PC metastasis. We found that HGF/c-Met inhibition reduced both the risk and rate of disease progression after resection and that this effect was associated with reduced cPSC counts. In conclusion, this study is the first to demonstrate the efficacy of adjuvant HGF/c-Met inhibition and is also the first to confirm the existence of cPSCs in PC.Background: Inhibition of hepatocyte growth factor (HGF)/c-MET pathway, a major mediator of pancreatic stellate cell (PSC)−PC cell interactions, retards local and distant cancer progression. This study examines the use of this treatment in preventing PC progression after resection. We further investigate the postulated existence of circulating PSCs (cPSCs) as a mediator of metastatic PC. Methods: Two orthotopic PC mouse models, produced by implantation of a mixture of luciferase-tagged human pancreatic cancer cells (AsPC-1), and human PSCs were used. Model 1 mice underwent distal pancreatectomy 3-weeks post-implantation (n = 62). One-week post-resection, mice were randomised to four treatments of 8 weeks: (i) IgG, (ii) gemcitabine (G), (iii) HGF/c-MET inhibition (HiCi) and (iv) HiCi + G. Tumour burden was assessed longitudinally by bioluminescence. Circulating tumour cells and cPSCs were enriched by filtration. Tumours of Model 2 mice progressed for 8 weeks prior to the collection of primary tumour, metastases and blood for single-cell RNA-sequencing (scRNA-seq). Results: HiCi treatments: (1) reduced both the risk and rate of disease progression after resection; (2) demonstrated an anti-angiogenic effect on immunohistochemistry; (3) reduced cPSC counts. cPSCs were identified using immunocytochemistry (α-smooth muscle actin+, pan-cytokeratin−, CD45−), and by specific PSC markers. scRNA-seq confirmed the existence of cPSCs and identified potential genes associated with development into cPSCs. Conclusions: This study is the first to demonstrate the efficacy of adjuvant HGF/c-Met inhibition for PC and provides the first confirmation of the existence of circulating PSCs.

Breaking the cycle: Targeting of NDRG1 to inhibit bi-directional oncogenic cross-talk between pancreatic cancer and stroma.

Pancreatic cancer (PaCa) is characterized by dense stroma that hinders treatment efficacy, with pancreatic stellate cells (PSCs) being a major contributor to this stromal barrier and PaCa progression. Activated PSCs release hepatocyte growth factor (HGF) and insulin-like growth factor (IGF-1) that induce PaCa proliferation, metastasis and resistance to chemotherapy. We demonstrate for the first time that the metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1), is a potent inhibitor of the PaCa-PSC cross-talk, leading to inhibition of HGF and IGF-1 signaling. NDRG1 also potently reduced the key driver of PaCa metastasis, namely GLI1, leading to reduced PSC-mediated cell migration. The novel clinically trialed anticancer agent, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), which upregulates NDRG1, potently de-sensitized PaCa cells to ligands secreted by activated PSCs. DpC and NDRG1 also inhibited the PaCa-mediated activation of PSCs via inhibition of sonic hedgehog (SHH) signaling. In vivo, DpC markedly reduced PaCa tumor growth and metastasis more avidly than the standard chemotherapy for this disease, gemcitabine. Uniquely, DpC was selectively cytotoxic against PaCa cells, while "re-programming" PSCs to an inactive state, decreasing collagen deposition and desmoplasia. Thus, targeting NDRG1 can effectively break the oncogenic cycle of PaCa-PSC bi-directional cross-talk to overcome PaCa desmoplasia and improve therapeutic outcomes.

Hepatocyte growth factor overexpression promotes osteoclastogenesis and exacerbates bone loss in CIA mice

BackgroundHepatocyte growth factor (HGF) is a multifunctional growth factor that promotes various biological processes. However, the effect of HGF on bone metabolism in rheumatoid arthritis (RA) remains unknown. Here, we investigated the role of HGF in regulating osteoclastogenesis and bone resorption in RA.MethodsThe expression of HGF in RA patients and collagen-induced arthritis (CIA) mice was examined. The role of HGF on osteoclastogenesis was analysed by osteoclastogenesis and bone resorption assays. The effect of HGF inhibition was evaluated in a CIA mice model. The mechanism of HGF in regulating osteoclastogenesis and bone resorption was explored by a series of in vitro studies.ResultsHGF was overexpressed in CIA and RA. HGF stimulated osteoclastogenesis in vitro. SU11274, a selective small molecule blocker of c-Met, impeded the effect of HGF on osteoclastogenesis and bone resorption. HGF regulated osteoclastogenesis by JNK and AKT-GSK-3β-NFATc1 signallings. SU11274 protected CIA mice from pathological bone loss.ConclusionsThese data strongly suggest that the highly expressed HGF in the joint tissues contributes to bone loss in RA. Inhibition of HGF/c-Met could effectively alleviate pathological bone loss and inflammatory symptoms in CIA mice. HGF/c-Met may be used as a new target for the treatment of bone loss in RA.

Abstract 1907: Src-Met signaling confers apoptosis resistance to EGFR and PI3K co-targeting independently of AKT and ERK pathway activation in head and neck cancer

Abstract Purpose: Molecularly targeted agents will play a major role in the next generation of personalized cancer therapies. However, intrinsic and acquired resistance to targeted agents poses challenges to the success of such treatments. The purpose of the current study was to investigate the intrinsic resistance of head and neck cancer (HNC) cell lines to apoptosis induced by the combination of EGFR inhibitor erlotinib and PI3K inhibitor BKM120. Methods: In a panel of 10 malignant and 1 premalignant HNC cell lines, we evaluated cell growth inhibition (by SRB assay), IC50, combination index, dose reduction index (by CalcuSyn Software), and apoptosis (by Annexin-V staining). Protein expression levels were measured by Western blotting. Small molecule inhibitors and siRNA-mediated knockdown strategies were used to inactivate and shut down the expression of the relevant proteins, respectively. Results: We have reported previously that single targeting of EGFR with erlotinib or PI3K with BKM120 (pan-PI3K inhibitor) suppressed cellular growth without inducing significant apoptosis. Co-targeting EGFR and PI3K demonstrated in vitro synergy in all except the JHU022 cell line and more effectively inhibited HNC xenograft growth in vivo. The combination of erlotinib and BKM120 induced variable apoptosis: some cell lines were very sensitive (MDA686TU, MDA686LN, 93-VU-147T), some moderately (Fadu, SqCCy1, MDA1483, UPCI:SCC090) and others were resistant (UD-SCC2, MSK-LEUK1, JHU022) to apoptosis induction. We found that the resistant cell line JHU022 expressed very high levels of p-Met (Y1234/1235). Inhibition of Met with small molecule inhibitor crizotinib or siRNA rendered this cell line highly sensitive to apoptosis induced by the combination of erlotinib and BKM120. Although inhibition of hepatocyte growth factor (HGF), the only known ligand for Met, with soluble neutralization antibody had no effect on Met phosphorylation, inhibition of Src family kinases (SFK) with dasatinib abolished Met phosphorylation at Y1234/1235. Dasatinib also made this cell line sensitive to apoptosis. Ablation of c-Src with siRNA had similar effects on p-Met (Y1234/1235) and apoptosis. Interestingly, inhibition of Met had no effects on AKT and ERK phosphorylation, two critical downstream pathways of receptor tyrosine kinases including Met. Conclusions: Our results strongly suggest that co-targeting of EGFR and PI3K has synergistic activity, is effective in vivo, and induces apoptosis of some HNC cell lines. Some resistant cell lines express a high level of p-Met as a downstream target of SFK, which confers resistance of these cells to EGFR and PI3K co-targeting. Finally, Met confers apoptosis resistance independently of AKT and ERK pathway activation. Citation Format: Abu S. Anisuzzaman, Abedul Haque, Zhuo G. Chen, Dong M. Shin, A.R.M. Ruhul Amin. Src-Met signaling confers apoptosis resistance to EGFR and PI3K co-targeting independently of AKT and ERK pathway activation in head and neck cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1907.

Targeting the HGF/c-MET pathway in advanced pancreatic cancer: a key element of treatment that limits primary tumour growth and eliminates metastasis

BackgroundStromal–tumour interactions facilitate pancreatic cancer (PC) progression. The hepatocyte growth factor (HGF)/c-MET pathway is upregulated in PC and mediates the interaction between cancer cells and stromal pancreatic stellate cells (PSCs). This study assessed the effect of HGF/c-MET inhibition plus gemcitabine (G) on the progression of advanced PC.MethodsOrthotopic PC was produced by implantation of luciferase-tagged human cancer cells + human PSCs into mouse pancreas. Tumours were allowed to develop without treatment for 4 weeks. Mice were then treated for 6 weeks with one of the following: IgG, G, HGF inhibitor (Hi), c-MET inhibitor (Ci), Hi + Ci, Hi + G, Ci + G, or Hi + Ci + G.ResultsBioluminescence imaging showed similar tumour sizes in all mice at the initiation of treatments. Triple therapy (Hi + Ci + G): (1) completely eliminated metastasis; (2) significantly reduced tumour size as assessed by bioluminescence and at necropsy; (3) significantly reduced proliferating cancer cell density and stem cell marker DCLK1 expression in tumours. In vitro 3D culture studies supported our in vivo findings.ConclusionEven at an advanced disease stage, a two-pronged approach, targeting (a) HGF/c-MET with relevant inhibitors and (b) cancer cells with chemotherapy, completely eliminated metastasis and significantly decreased tumour growth, suggesting that this is a promising treatment approach for PC.

Resveratrol Suppresses Prostate Cancer Epithelial Cell Scatter/Invasion by Targeting Inhibition of Hepatocyte Growth Factor (HGF) Secretion by Prostate Stromal Cells and Upregulation of E-cadherin by Prostate Cancer Epithelial Cells.

Cancer mortality is primarily attributed to metastasis and the resulting compromise of organs secondary to the initial tumor site. Metastasis is a multi-step process in which the tumor cells must first acquire a migratory phenotype and invade through the surrounding tissue for spread to distant organs in the body. The ability of malignant cells to migrate and breach surrounding tissue/matrix barriers is among the most daunting challenges to disease management for men in the United States diagnosed with prostate cancer (CaP), especially since, at diagnosis, a high proportion of patients already have occult or clinically-detectable metastasis. The interaction between hepatocyte growth factor (HGF) secreted by the stroma, with its receptor c-Met located in the epithelium, must occur for epithelial CaP cells to become migratory. We studied the effects of grape-derived phytochemical resveratrol on the transition of epithelial tumor cells from sedentary to a mobile, penetrant phenotype. A time lapse microscopy assay was used to monitor the acquisition of the migratory phenotype by resveratrol. The results show that resveratrol inhibits HGF-mediated interaction between the stroma and epithelium and suppresses epithelial CaP cell migration by attenuating the control of epithelial-to-mesenchymal transition (EMT).

Macrophage HIF-2α regulates tumor-suppressive Spint1 in the tumor microenvironment.

In solid tumors, tumor-associated macrophages (TAMs) commonly accumulate within hypoxic areas. Adaptations to such environments evoke transcriptional changes by the hypoxia-inducible factors (HIFs). While HIF-1α is ubiquitously expressed, HIF-2α appears tissue-specific with consequences of HIF-2α expression in TAMs only being poorly characterized. An E0771 allograft breast tumor model revealed faster tumor growth in myeloid HIF-2α knockout (HIF-2αLysM-/- ) compared with wildtype (wt) mice. In an RNA-sequencing approach of FACS sorted wt and HIF-2α LysM-/- TAMs, serine protease inhibitor, Kunitz type-1 ( Spint1) emerged as a promising candidate for HIF-2α-dependent regulation. We validated reduced Spint1 messenger RNA expression and concomitant Spint1 protein secretion under hypoxia in HIF-2α-deficient bone marrow-derived macrophages (BMDMs) compared with wt BMDMs. In line with the physiological function of Spint1 as an inhibitor of hepatocyte growth factor (HGF) activation, supernatants of hypoxic HIF-2α knockout BMDMs, not containing Spint1, were able to release proliferative properties of inactive pro-HGF on breast tumor cells. In contrast, hypoxic wt BMDM supernatants containing abundant Spint1 amounts failed to do so. We propose that Spint1 contributes to the tumor-suppressive function of HIF-2α in TAMs in breast tumor development.

M2 macrophages mediate sorafenib resistance by secreting HGF in a feed-forward manner in hepatocellular carcinoma

BackgroundSorafenib is the only approved first line systemic therapy for advanced hepatocellular carcinoma (HCC) in the last decade. Tumour resistance to sorafenib has been of major obstacles to improve HCC patient survival.MethodsWe polarised THP-1 cells to M1 and M2 macrophages, performed various in vitro assays and developed sorafenib-resistant xenograft models to investigate the role of tumour-associated macrophages (TAM)-secreted molecules in HCC resistance to the targeted therapy.ResultsWe demonstrated M2, but not M1, macrophages not only promote proliferation, colony formation and migration of hepatoma cells but also significantly confer tumour resistance to sorafenib via sustaining tumour growth and metastasis by secreting hepatocyte growth factor (HGF). HGF activates HGF/c-Met, ERK1/2/MAPK and PI3K/AKT pathways in tumour cells. Tumour-associated M2 macrophages were accumulated in sorafenib-resistance tumours more than in sorafenib-sensitive tumours in vivo and produced abundant HGF. HGF chemoattracts more macrophages migrated from surrounding area, regulates the distribution of M2 macrophages and increases hepatoma resistance to sorafenib in a feed-forward manner.ConclusionsOur results provide new insights into the mechanisms of sorafenib resistance in HCC and rationale for developing new trials by combining sorafenib with a potent HGF inhibitor such as cabozantinib to improve the first line systemic therapeutic efficacy.

FOLFOX alone or combined with rilotumumab or panitumumab as first-line treatment for patients with advanced gastroesophageal adenocarcinoma (PRODIGE 17-ACCORD 20-MEGA): a randomised, open-label, three-arm phase II trial

BackgroundEpidermal growth factor receptor (EGFR) and hepatocyte growth factor (HGF)/mesenchymal–epithelial transition (MET) pathways, which promote tumour growth and proliferation, are often deregulated in advanced gastroesophageal adenocarcinomas. We assessed whether adding panitumumab (an EGFR inhibitor) or rilotumumab (a HGF inhibitor) to first-line fluoropyrimidine-based and platinum-based chemotherapy (modified oxaliplatin, leucovorin and fluorouracil [mFOLFOX6]) benefits to patients with advanced gastroesophageal adenocarcinoma. Patients and methodsThis phase II, open-label, randomised, three-arm study enrolled patients ≥18 years, with advanced gastroesophageal adenocarcinoma, Eastern Cooperative Oncology Group performance status 0–1 and no known HER2 overexpression. Patients were randomly assigned (1:1:1) mFOLFOX6 (oxaliplatin 85 mg/m2, leucovorin 400 mg/m2, 5-fluorouracil 400 mg/m2 bolus then 2400 mg/m2 over 46 h) alone or combined with panitumumab (6 mg/kg) or rilotumumab (10 mg/kg) every 2 weeks until limiting toxicity, patient's refusal or disease progression. The primary end-point was the 4-month progression-free survival (PFS) rate. Secondary end-points included overall survival (OS) and tolerance. ResultsThe study enrolled 162 patients in 29 French centres. The median follow-up was 23.6 months (interquartile range = 16.4–29.0). The 4-month PFS rate was 71% (95% confidence interval [CI] = 57–82) with chemotherapy alone, 57% (95% CI = 42–71) combined with panitumumab and 61% (95% CI = 47–74) combined with rilotumumab. Median OS was 13.1 months (95% CI = 8.7–16.9) with chemotherapy alone, 8.3 months (95% CI = 6.2–13.2) combined with panitumumab and 11.5 months (95% CI = 7.9–17.1) combined with rilotumumab. Adverse events grade ≥III occurred less frequently with chemotherapy alone (62%) than with panitumumab (83%) and rilotumumab (89%). ConclusionsWe found no benefit in adding panitumumab or rilotumumab to mFOLFOX6 first-line chemotherapy to treat advanced gastroesophageal adenocarcinoma patients. Trial registrationEuropean Clinical Trials Database, number 2009-012797-12.

Bone Metastasis Phenotype and Growth Undergo Regulation by Micro-Environment Stimuli: Efficacy of Early Therapy with HGF or TGFβ1-Type I Receptor Blockade.

Hepatocyte growth factor (HGF) and transforming growth factor β1 (TGFβ1) are biological stimuli of the micro-environment which affect bone metastasis phenotype through transcription factors, but their influence on the growth is scarcely known. In a xenograft model prepared with 1833 bone metastatic cells, derived from breast carcinoma cells, we evaluated mice survival and Twist and Snail expression and localization after competitive inhibition of HGF with NK4, or after blockade of TGFβ1-type I receptor (RI) with SB431542: in the latter condition HGF was also measured. To explain the in vivo data, in 1833 cells treated with SB431542 plus TGFβ1 we measured HGF formation and the transduction pathway involved. Altogether, HGF seemed relevant for bone-metastatic growth, being hampered by NK4 treatment, which decreased Twist more than Snail in the metastasis bulk. TGFβ1-RI blockade enhanced HGF in metastasis and adjacent bone marrow, while reducing prevalently Snail expression at the front and bulk of bone metastasis. The HGF accumulation in 1833 cells depended on an auxiliary signaling pathway, triggered by TGFβ1 under SB431542, which interfered in the transcription of HGF activator inhibitor type 1 (HAI-1) downstream of TGFβ-activated kinase 1 (TAK1): HGF stimulated Twist transactivation. In conclusion, the impairment of initial outgrowth with NK4 seemed therapeutically promising more than SB431542 chemotherapy; a functional correlation between Twist and Snail in bone metastasis seemed to be influenced by the biological stimuli of the micro-environment, and the targeting of these phenotype biomarkers might inhibit metastasis plasticity and colonization, even if it would be necessary to consider the changes of HGF levels in bone metastases undergoing TGFβ1-RI blockade.

HGF regulate HTR-8/SVneo trophoblastic cells migration/invasion under hypoxic conditions through increased HIF-1α expression via MAPK and PI3K pathways.

Hepatocyte growth factor (HGF) is reported to be down-regulated in pregnancy complications like intrauterine growth retardation and preeclampsia, which are associated with abnormal trophoblast migration/invasion. In this study, role of HGF and associated signaling pathways has been investigated in HTR-8/SVneo trophoblastic cells migration/invasion under normoxia (20% O2) and hypoxia (2% O2). HTR-8/SVneo cells exposed to hypoxia showed increase in migration and invasion as compared to cells incubated under normoxic conditions. The migration/invasion under both normoxic and hypoxic conditions was further enhanced after treatment with HGF. Subsequent to treatment with HGF, a significant increase in expression of MMP2 & MMP3 under normoxia and MMP1 & MMP9 under hypoxia was observed. Treatment of HTR-8/SVneo cells with HGF under hypoxia also led to decrease in TIMP1. Treatment of the cells with HGF led to activation of mitogen activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. Inhibition of MAPK by U0126 and PI3K by LY294002 led to concomitant decrease in the HGF-mediated migration/invasion of HTR-8/SVneo cells. HGF treatment under hypoxia also led to a significant increase in hypoxia inducible factor (HIF-1α) expression. Additionally, inhibition of HIF-1α by siRNA led to decrease in HGF-mediated migration of HTR-8/SVneo cells under hypoxic conditions. Inhibition of HGF activated MAPK and PI3K signaling led to reduction in HIF-1α expression under hypoxia. In conclusion, HGF facilitates HTR-8/SVneo cell migration/invasion by activation of MAPK/PI3K signaling pathways and increased expression of MMPs. HIF-1α has a role in HGF-mediated increase in migration under hypoxic conditions.

Inhibition of hepatocyte growth factor on proliferation and transdifferentiation of human Tenon capsule fibroblasts triggered by transforming growth factor-β1in vitro

Objective To investigate the effects of hepatocyte growth factor (HGF) on proliferation and transdifferentiation of human Tenon capsule fibroblasts induced by transforming growth factor -β1 (TGF-β1) in vitro. Methods Human Tenon capsule fibroblasts were cultured and divided into blank control group, TGF-β1 treated group and different concentrations HGF+ TGF-β1 groups.The TGF-β1 at 10 μg/L was added into culture medium of the TGF-β1 treated group, and different concentrations of HGF (25, 50, 100, 200 μg/L) and 10 μg/L TGF-β1 were added into culture medium of the HGF25 μg/L+ TGF-β1, HGF50 μg/L+ TGF-β1, HGF100 μg/L+ TGF-β1, HGF200 μg/L+ TGF-β1 group respectively, Methyl thiazolyl tetrazolium (MTT) was employed to measure the cell proliferation (absorbance at 560 nm). Immunofluorescence staining was used to evaluate and locate the expression of α-smooth muscle action (α-SMA) in the cells.The expression of α-SMA protein in the cells was detected by Western blot assay. Results Cultured cells showed fusiform in shape with the positive response for vimentin.The proliferation value of the cells was 0.203±0.025, 0.497±0.101, 0.426±0.062, 0.354±0.040, 0.272±0.084, 0.241±0.011 in the blank control group, TGF-β1 treated group, HGF25 μg/L+ TGF-β1 group, HGF50 μg/L+ TGF-β1 group, HGF100 μg/L+ TGF-β1 group and HGF200 μg/L+ TGF-β1 group, respectively, showing a significant difference among the groups (F=9.210, P=0.003). Compared with the TGF-β1 treated group, the proliferation values of the cells were significantly reduced in the blank control group and HGF+ TGF-β1 group (all at P<0.05). Immunofluorescence staining showed that α-SMA protein mainly located in cytoplasm with the strong red fluorescence in the cells of the TGF-β1 treated group and weak red fluorescence in HGF+ TGF-β1 group, and the expression of α-SMA was absent in the blank control group.The percentage of α-SMA-positive cells was (60.0±4.7)% in the TGF-β1 treated group and (14.3±3.1)% in the HGF+ TGF-β1 group, with significant difference between the two groups (t=19.856, P<0.001). The relative expression levels of the α-SMA protein in the cells were 0.642±0.032, 1.330±0.069 and 0.884±0.040 in the blank control group, TGF-β1 group and HGF100 μg/L+ TGF-β1 group, respectively, showing a significant difference among the groups (F=13.370, P<0.001), and relative expression levels of the α-SMA protein in the cells were significantly lower in the blank control group and HGF100 μg/L+ TGF-β1 group than that in the TGF-β1 treated group (all at P<0.05). Conclusions HGF can inhibit the proliferation of human Tenon capsule fibroblasts, down-regulate the expression of α-SMA protein induced by TGF-β1 and arrest the phenotype transformation of fibroblasts in vitro. Key words: Tenon capsule/cytology; Fibroblasts/metabolism; Hepatocyte growth factor/administration & dosage; Transforming growth factor beta type 1; Fibrosis/prevention & control; α-Smooth muscular actin; Humans; Cells, cultured

The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts

BackgroundAdipose tissue is an excellent source for isolation of stem cells for treating various clinical conditions including injuries to the neuromuscular system. Many previous studies have focused on differentiating these adipose stem cells (ASCs) towards a Schwann cell-like phenotype (dASCs), which can enhance axon regeneration and reduce muscle atrophy. However, the stromal vascular fraction (SVF), from which the ASCs are derived, also exerts broad regenerative potential and might provide a faster route to clinical translation of the cell therapies for treatment of neuromuscular disorders.MethodsThe aim of this study was to establish the effects of SVF cells on the proliferation and differentiation of myoblasts using indirect co-culture experiments. A Growth Factor PCR Array was used to compare the secretomes of SVF and dASCs, and the downstream signaling pathways were investigated.ResultsSVF cells, unlike culture-expanded dASCs, expressed and secreted hepatocyte growth factor (HGF) at concentrations sufficient to enhance the proliferation of myoblasts. Pharmacological inhibitor studies revealed that the signal is mediated via ERK1/2 phosphorylation and that the effect is significantly reduced by the addition of 100 pM Norleual, a specific HGF inhibitor. When myoblasts were differentiated into multinucleated myotubes, the SVF cells reduced the expression levels of fast-type myosin heavy chain (MyHC2) suggesting an inhibition of the differentiation process.ConclusionsIn summary, this study shows the importance of HGF as a mediator of the SVF effects on myoblasts and provides further evidence for the importance of the secretome in cell therapy and regenerative medicine applications.

Up-regulation of SPINT2/HAI-2 by Azacytidine in bone marrow mesenchymal stromal cells affects leukemic stem cell survival and adhesion.

The role of tumour microenvironment in neoplasm initiation and malignant evolution has been increasingly recognized. However, the bone marrow mesenchymal stromal cell (BMMSC) contribution to disease progression remains poorly explored. We previously reported that the expression of serine protease inhibitor kunitz‐type2 (SPINT2/HAI‐2), an inhibitor of hepatocyte growth factor (HGF) activation, is significantly lower in BMMSC from myelodysplastic syndromes (MDS) patients compared to healthy donors (HD). Thus, to investigate whether this loss of expression was due to SPINT2/HAI‐2 methylation, BMMSC from MDS and de novo acute myeloid leukaemia (de novo AML) patients were treated with 5‐Azacitidine (Aza), a DNA methyltransferase inhibitor. In MDS‐ and de novo AML‐BMMSC, Aza treatment resulted in a pronounced SPINT2/HAI‐2 levels up‐regulation. Moreover, Aza treatment of HD‐BMMSC did not improve SPINT2/HAI‐2 levels. To understand the role of SPINT2/HAI‐2 down‐regulation in BMMSC physiology, SPINT2/HAI‐2 expression was inhibited by lentivirus. SPINT2 underexpression resulted in an increased production of HGF by HS‐5 stromal cells and improved survival of CD34+ de novo AML cells. We also observed an increased adhesion of de novo AML hematopoietic cells to SPINT2/HAI‐2 silenced cells. Interestingly, BMMSC isolated from MDS and de novo AML patients had increased expression of the integrins CD49b, CD49d, and CD49e. Thus, SPINT2/HAI‐2 may contribute to functional and morphological abnormalities of the microenvironment niche and to stem/progenitor cancer cell progression. Hence, down‐regulation in SPINT2/HAI‐2 gene expression, due to methylation in MDS‐BMMSC and de novo AML‐BMMSC, provides novel insights into the pathogenic role of the leukemic bone marrow microenvironment.

Targeting the HGF/c-MET pathway: stromal remodelling in pancreatic cancer.

Stromal-tumor interactions in pancreatic cancer (PC) impact on treatment outcomes. Pancreatic stellate cells (PSCs) produce the collagenous stroma of PC and interact with cancer cells to facilitate disease progression. A candidate growth factor pathway that may mediate this interaction is the hepatocyte growth factor (HGF)/c-MET pathway. HGF is produced by PSCs and its receptor c-MET is expressed on pancreatic cancer cells. We studied the effects on PC progression of inhibiting the HGF/c-MET pathway in the presence and absence of a representative chemotherapeutic agent, gemcitabine. Using an orthotopic model of PC we have shown that “triple therapy” (inhibition of both HGF and c-MET combined with gemcitabine) resulted in the greatest reduction in tumor volume compared to each of the treatments alone or in dual combinations. Importantly, metastasis was virtually eliminated in mice receiving triple therapy. Our in vivo findings were supported by in vitro studies showing that the increase in cancer cell proliferation and migration in response to PSC secretions was significantly inhibited by the triple regimen. Our studies suggest that a combined approach, that targets tumor cells by chemotherapy while inhibiting specific pathways that mediate stromal-tumor interactions, may represent a novel therapeutic strategy to improve outcomes in PC.

Endoplasmic reticulum stress restrains hepatocyte growth factor expression in hepatic stellate cells and rat acute liver failure model

The aim of this study was to test whether endoplasmic reticulum (ER) stress suppresses hepatocyte growth factor (HGF) expression in hepatic stellate cells (HSCs) and whether ER stress plays a role in alleviating d-Galactosamine and Lipopolysaccharide (D-GalN/LPS)-induced acute liver failure (ALF) by regulating HGF expression. Rat HSCs line HSC-T6 were treated with the ER stress inducers tunicamycin or thapsigargin, and ER stress inhibitors sodium 4-phenylbutyrate (4-PBA) or salubrinal, respectively. Recombinant lentivirus containing eukaryotic translation initiation factor 2α (eIf2α), named LV-eIf2α-shRNA-GFP was produced to block eIf2α activated by ER stress. A rat ALF model was created by intraperitoneal injection of D-GalN/LPS, and 100 mg/kg 4-PBA was injected 6 h before injection as an inhibitor of ER stress. Levels of HGF, c-Met, vascular endothelial growth factor (VEGF), GRP78, eIf2α, phospho-eIF2α, ATF4 and CHOP in vitro and in vivo were measured. Our results demonstrated that ER stress stimulated VEGF but inhibited HGF expression in HSC-T6 cells. Both the stimulation of VEGF and the inhibition of HGF could be partly prevented by 4-PBA or Salubrinal. eIf2α expression was upregulated during ERS and interfering eIf2α expression proportionately down-regulated HGF expression. Inhibition of HGF and c-Met expression was also observed in the ALF rat. Treatment with 4-PBA prevented the reduction of HGF in ALF rats. ER stress regulates HGF expression in vitro and in vivo, which depends on eIf2α pathway. Reduction in liver damage of ALF by 4-PBA is associated with attenuation of ER stress and maintaining HGF production.

Analogs of the hepatocyte growth factor and macrophage-stimulating protein hinge regions act as Met and Ron dual inhibitors in pancreatic cancer cells

Pancreatic cancer is among the leading causes of cancer death in the USA, with limited effective treatment options. A major contributor toward the formation and persistence of pancreatic cancer is the dysregulation of the hepatocyte growth factor (HGF)/Met (HGF receptor) and the macrophage-stimulating protein (MSP)/Ron (MSP receptor) systems. These systems normally mediate a variety of cellular behaviors including proliferation, survival, and migration, but are often overactivated in pancreatic cancer and contribute toward cancer progression. Previous studies have shown that HGF must dimerize to activate Met. Small-molecule antagonists with homology to a 'hinge' region within the putative dimerization domain of HGF have been developed that bind to HGF and block dimerization, therefore inhibiting Met signaling. Because of the structural and sequence homology between MSP and HGF, we hypothesized that the inhibition of HGF by the hinge analogs may extend to MSP. The primary aim of this 'proof-of-concept' study was to determine whether hinge analogs could inhibit cellular responses to both HGF and MSP in pancreatic cancer cells. Our results showed that these compounds inhibited HGF and MSP activity. Hinge analog treatment resulted in decreased Met and Ron activation, and suppressed malignant cell behaviors including proliferation, migration, and invasion in pancreatic cancer cells in vitro. These results suggest that the hinge analogs represent a novel group of molecules that may offer a therapeutic approach for the treatment of pancreatic cancer and warrant further development and optimization.

Effect of brefelamide on HGF-induced survival of 1321N1 human astrocytoma cells.

Malignant gliomas are characterized by their high level of resistance to chemo- and radiotherapy and new treatment options are urgently required. We previously demonstrated that brefelamide, an aromatic amide isolated from methanol extracts of cellular slime molds Dictyostelium brefeldianum and D. giganteum, had antiproliferative effects on 1321N1 human astrocytoma cells, a model of glioma. In this study, we investigated the mechanisms by which brefelamide inhibited 1321N1 and PC12 rat pheochromocytoma cell proliferation. When cells were cultured in serum-free medium, hepatocyte growth factor (HGF) increased survival of 1321N1 cells but not PC12 cells. HGF receptor, c-MET, was strongly expressed in 1321N1 cells, but not in PC12 cells. Pretreatment of 1321N1 cells with brefelamide inhibited both HGF-induced cell survival and expression of c-MET. Phosphorylation of extracellular signal-regulated kinase (ERK) and AKT was increased by HGF, but these changes were inhibited by brefelamide pretreatment. Moreover, HGF mRNA levels and secretion were reduced by brefelamide. These results suggest that brefelamide reduces survival of 1321N1 cells via multiple effects including suppression of HGF receptor expression and HGF secretion and inhibition of ERK and AKT phosphorylation.