Secretion Of Vascular Endothelial Growth factor-C Research Articles

DHCR7 promotes lymph node metastasis in cervical cancer through cholesterol reprogramming-mediated activation of the KANK4/PI3K/AKT axis and VEGF-C secretion

Cervical cancer (CC) patients with lymph node metastasis (LNM) have a poor prognosis. However, the molecular mechanism of LNM in CC is unclear, and there is no effective clinical treatment. Here, we found that 7-dehydrocholesterol reductase (DHCR7), an enzyme that catalyzes the last step of cholesterol synthesis, was upregulated in CC and closely related to LNM. Gain-of-function and loss-of-function experiments proved that DHCR7 promoted the invasion ability of CC cells and lymphangiogenesis in vitro and induced LNM in vivo. The LNM-promoting effect of DHCR7 was partly mediated by upregulating KN motif and ankyrin repeat domains 4 (KANK4) expression and subsequently activating the PI3K/AKT signaling pathway. Alternatively, DHCR7 promoted the secretion of vascular endothelial growth factor-C (VEGF-C), and thereby lymphangiogenesis. Interestingly, cholesterol reprogramming was needed for the DHCR7-mediated promotion of activation of the KANK4/PI3K/AKT axis, VEGF-C secretion, and subsequent LNM. Importantly, treatment with the DHCR7 inhibitors AY9944 and tamoxifen (TAM) significantly inhibited LNM of CC, suggesting the clinical application potential of DHCR7 inhibitors in CC. Collectively, our results uncover a novel molecular mechanism of LNM in CC and identify DHCR7 as a new potential therapeutic target.

Macrophage-produced VEGFC is induced by efferocytosis to ameliorate cardiac injury and inflammation.

Clearance of dying cells by efferocytosis is necessary for cardiac repair after myocardial infarction (MI). Recent reports have suggested a protective role for vascular endothelial growth factor C (VEGFC) during acute cardiac lymphangiogenesis after MI. Here, we report that defective efferocytosis by macrophages after experimental MI led to a reduction in cardiac lymphangiogenesis and Vegfc expression. Cell-intrinsic evidence for efferocytic induction of Vegfc was revealed after adding apoptotic cells to cultured primary macrophages, which subsequently triggered Vegfc transcription and VEGFC secretion. Similarly, cardiac macrophages elevated Vegfc expression levels after MI, and mice deficient for myeloid Vegfc exhibited impaired ventricular contractility, adverse tissue remodeling, and reduced lymphangiogenesis. These results were observed in mouse models of permanent coronary occlusion and clinically relevant ischemia and reperfusion. Interestingly, myeloid Vegfc deficiency also led to increases in acute infarct size, prior to the amplitude of the acute cardiac lymphangiogenesis response. RNA-Seq and cardiac flow cytometry revealed that myeloid Vegfc deficiency was also characterized by a defective inflammatory response, and macrophage-produced VEGFC was directly effective at suppressing proinflammatory macrophage activation. Taken together, our findings indicate that cardiac macrophages promote healing through the promotion of myocardial lymphangiogenesis and the suppression of inflammatory cytokines.

Qingjie Fuzheng Granule suppresses lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway

ScopeTo investigate the effect of Qingjie Fuzheng Granule (QFG) on lymphangiogenesis and lymphatic metastasis in colorectal cancer. MethodsThe effects of QFG on the expression and secretion of vascular endothelial growth factor-C (VEGF-C) in HCT-116 cells were investigated both in vitro and in vivo. HCT-116 cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. The VEGF-C expression level was determined using RT-qPCR and western blotting, and the VEGF-C concentration in supernatant was measured by ELISA. Tumor xenograft models of HCT-116 cells were generated using BALB/c nude mice, and the mice were randomly divided into a control group (gavaged with normal saline) and QFG group (gavaged with 2 g/kg QFG). The effect of QFG on tumor growth was evaluated by comparing the volume and weight of tumors between two groups. Immunohistochemistry (IHC) and RT-qPCR were performed to detect the expression levels of VEGF-C, vascular endothelial growth factor receptor 3 (VEGFR-3), and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1). ELISA was performed to measure the concentration of serum VEGF-C. TMT proteomics technology and Reactome pathway analysis were used to explore the mechanism of QFG inhibiting lymphangiogenesis in tumor. The VEGF-C (5 ng/mL)-stimulated human lymphatic endothelial cell (HLEC) model was conducted to evaluate the effect of QFG on lymphangiogenesis in vitro. The model cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. Cell viability was then determined using an MTT assay. The cell migration, invasion, and tube-formation ability were analyzed using transwell migration, matrigel invasion and tube formation assays, respectively. The underlying mechanism was uncovered, the levels of VEGFR-3, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), p-PI3K/PI3K, p-AKT/AKT and p-mTOR/ mTOR were detected using western blotting. ResultsQFG significantly reduced VEGF-C expression and secretion in HCT-116 cells. QFG evidently suppressed in vivo tumor growth and the expression of VEGF-C, VEGFR-3, and LYVE-1. The serum VEGF-C level was also reduced by QFG. Moreover, TMT proteomics technology and Reactome pathway analysis identified 95 differentially expressed protein and multiple enriched pathway about matrix metalloproteinase and extracellular matrix, which is direct associate with lymphangiogenesis. In vitro experiment, QFG inhibited the viability, migration, invasion and tube formation of HLECs. Additionally, QFG reduced the VEGFR-3, MMP-2, MMP-9 expression levels, and the p-PI3K/PI3K, p-AKT/AKT, p-mTOR/ mTOR ratios. ConclusionQFG can exert its effect on both tumor cells and HLECs, exhibiting ani- lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway pathway.

DUSP2 regulates extracellular vesicle-VEGF-C secretion and pancreatic cancer early dissemination

ABSTRACT Early dissemination is a unique characteristic and a detrimental process of pancreatic ductal adenocarcinoma (PDAC); however, the underlying mechanism remains largely unknown. Here, we investigate the role of dual-specificity phosphatase-2 (DUSP2)-vascular endothelial growth factor-C (VEGF-C) axis in mediating PDAC lymphangiogenesis and lymphovascular invasion. Expression of DUSP2 is greatly suppressed in PDAC, which results in increased aberrant expression of extracellular vesicle (EV)-associated VEGF-C secretion. EV-VEGF-C exerts paracrine effects on lymphatic endothelial cells and autocrine effects on cancer cells, resulting in the lymphovascular invasion of cancer cells. Tissue-specific knockout of Dusp2 in mouse pancreas recapitulates PDAC phenotype and lymphovascular invasion. Mechanistically, loss-of-DUSP2 enhances proprotein convertase activity and vesicle trafficking to promote the release of the mature form of EV-VEGF-C. Collectively, these findings represent a conceptual advance in understanding pancreatic cancer lymphovascular invasion and suggest that loss-of-DUSP2-mediated VEGF-C processing may play important roles in early dissemination of pancreatic cancer. Abbreviations: DUSP2: dual-specificity phosphatase-2; VEGF-C: vascular endothelial growth factor-C; EV: extracellular vesicles; PDAC: pancreatic ductal adenocarcinoma; KD: knockdown

Heparanase-1-induced shedding of heparan sulfate from syndecan-1 in hepatocarcinoma cell facilitates lymphatic endothelial cell proliferation via VEGF-C/ERK pathway

Heparanase-1/syndecan-1 axis plays critical roles in tumorigenesis and development. The main mechanism includes heparanase-1 (HPA-1) degrades the heparan sulfate chain of syndecan-1 (SDC-1), and the following shedding of heparan sulfate from tumor cell releases and activates SDC-1 sequestered growth factors. However, the significance of Heparanase-1/syndecan-1 axis and its effects on the microenvironment of lymphatic metastasis in hepatocellular carcinogenesis (HCC) procession have not been reported. Herein, we found that HPA-1 could degrade the heparan sulfate on hepatocarcinoma cell surface. Importantly, HPA-1-induced shedding of heparan sulfate chain from SDC-1 facilitated the release of vascular endothelial growth factor C (VEGF-C) from SDC-1/VEGF-C complex into the medium of hepatocarcinoma cell. Further studies indicated that VEGF-C secretion from hepatocarcinoma cell promoted lymphatic endothelial cell growth through activating extracellular signal-regulated kinase (ERK) signaling. Taken together, this study reveals a novel existence of Heparanase-1/syndecan-1 axis in hepatocarcinoma cell and its roles in the cross-talking with the microenvironment of lymphatic metastasis.

Adiponectin promotes VEGF-C-dependent lymphangiogenesis by inhibiting miR-27b through a CaMKII/AMPK/p38 signaling pathway in human chondrosarcoma cells.

Chondrosarcoma is the second most frequently occurring typeof bone malignancy characterized by distant metastatic propensity. Vascular endothelial growth factor-C (VEGF-C) is the major lymphangiogenic factor, and makes crucial contributions to tumour lymphangiogenesis and lymphatic metastasis. Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes. In recent years, adiponectin has also been indicated as facilitating tumorigenesis, angiogenesis and metastasis. However, the effect of adiponectin on VEGF-C regulation and lymphangiogenesis in chondrosarcoma has remained largely a mystery. In the present study, we have shown a clinical correlation between adiponectin and VEGF-C, as well as tumour stage, in human chondrosarcoma tissues. We further demonstrated that adiponectin promoted VEGF-C expression and secretion in human chondrosarcoma cells. The conditioned medium from adiponectin-treated cells significantly induced tube formation and migration of human lymphatic endothelial cells. In addition, adiponectin knock down inhibited lymphangiogenesis invitro and invivo We also found that adiponectin-induced VEGF-C is mediated by the calmodulin-dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK) and p38 signaling pathway. Furthermore, the expression of miR-27b was negatively regulated by adiponectin via the CaMKII, AMPK and p38 cascade. The present study is the first to describe the mechanism of adiponectin-promoted lymphangiogenesis by up-regulating VEGF-C expression in chondrosarcomas. Thus, adiponectin could serve as a therapeutic target in chondrosarcoma metastasis and lymphangiogenesis.

Leptin promotes VEGF-C production and induces lymphangiogenesis by suppressing miR-27b in human chondrosarcoma cells.

Chondrosarcoma is the second most frequently occurring type of bone malignancy that is characterized by the distant metastasis propensity. Vascular endothelial growth factor-C (VEGF-C) is the chief lymphangiogenic mediator, and makes crucial contributions to tumor lymphangiogenesis. Leptin is an adipocytokine and has been indicated to facilitate tumorigenesis, angiogenesis and metastasis. However, the effect of leptin on VEGF-C regulation and lymphangiogenesis in human chondrosarcoma has hugely remained a mystery. Our results showed a clinical correlation between leptin and VEGF-C as well as tumor stage in human chondrosarcoma tissues. We further demonstrated that leptin promoted VEGF-C production and secretion in human chondrosarcoma cells. The conditioned medium from leptin-treated chondrosarcoma cells induced lymphangiogenesis of human lymphatic endothelial cells. We also found that leptin-induced VEGF-C is mediated by the FAK, PI3K and Akt signaling pathway. Furthermore, the expression of microRNA-27b was negatively regulated by leptin via the FAK, PI3K and Akt cascade. Our study is the first to describe the mechanism of leptin-promoted lymphangiogenesis by upregulating VEGF-C expression in chondrosarcomas. Thus, leptin could serve as a therapeutic target in chondrosarcoma metastasis and lymphangiogenesis.

CCL5 promotes VEGF-C production and induces lymphangiogenesis by suppressing miR-507 in human chondrosarcoma cells.

Chondrosarcoma is the second most frequently occurring type of bone malignancy that is characterized by the distant metastasis propensity. Vascular endothelial growth factor-C (VEGF-C) is the major lymphangiogenic factor, and makes crucial contributions to tumor lymphangiogenesis and lymphatic metastasis. Chemokine CCL5 has been reported to facilitate angiogenesis and metastasis in chondrosarcoma. However, the effect of chemokine CCL5 on VEGF-C regulation and lymphangiogenesis in chondrosarcoma has largely remained a mystery. In this study, we showed a clinical correlation between CCL5 and VEGF-C as well as tumor stage in human chondrosarcoma tissues. We further demonstrated that CCL5 promoted VEGF-C expression and secretion in human chondrosarcoma cells. The conditioned medium (CM) from CCL5-overexpressed cells significantly induced tube formation of human lymphatic endothelial cells (LECs). Mechanistic investigations showed that CCL5 activated VEGF-C-dependent lymphangiogenesis by down-regulating miR-507. Moreover, inhibiting CCL5 dramatically reduced VEGF-C and lymphangiogenesis in the chondrosarcoma xenograft animal model. Collectively, we document for the first time that CCL5 induces tumor lymphangiogenesis by the induction of VEGF-C in human cancer cells. Our present study reveals miR-507/VEGF-C signaling as a novel mechanism in CCL5-mediated tumor lymphangiogenesis. Targeting both CCL5 and VEGF-C pathways might serve as the potential therapeutic strategy to block cancer progression and metastasis in chondrosarcoma.

Influence of Photodynamic Therapy on Apoptosis and Invasion of Human Cholangiocarcinoma QBC939 Cell Line.

To investigate the effect of photodynamic therapy (PDT) mediated by hematoporphyrin derivative (HPD) on apoptosis and invasion of cholangiocarcinoma QBC939 cell lines. In vitro cultured cholangiocarcinoma QBC939 cell line was exposed to 2, 4, 6, 8, 10, 12, and 14 μg/ml HPD with 5, 10, and 15 J/cm2 light intensity, respectively. The optical density at 450 nm of the QBC939 cells was measured by CCK8 assay and its growth inhibition ratio was calculated. Flow cytometry and transwell migration assay were applied to detect cell apoptosis and invasion respectively. RT-PCR and immunocytochemistry analyses were used to detect expressions of vascular endothelial growth factor-C (VEGF-C), cyclooxygenase-2 (COX-2), and proliferating cell nuclear antigen (PCNA). Enzyme-linked immunosorbent assay (ELISA) was carried out to examine the secretion of VEGF-C and COX-2 in QBC939 cells. Exposure to HPD-PDT can significantly suppress the growth of QBC939 cells (all P<0.05). HPD-PDT can promote apoptosis of QBC939 cells at the early stage. When the concentration of HPD was 2 μg/ml and light irradiation was 5 J/cm2, HPD-PDT had no obvious inhibitory effect on QBC939 cell growth, but can obviously inhibit cell invasion, and significant difference was observed between the HPD-PDT and control groups (P<0.01). The HPD-PDT can reduce the mRNA and protein expressions of VEGF-C, COX-2, and PCNA, and decrease the secretion of VEGF-C and COX-2 in QBC939 cells. PDT could promote apoptosis and inhibit growth and invasion of cholangiocarcinoma cells QBC939 in vitro.

MicroRNA-27b, microRNA-101 and microRNA-128 inhibit angiogenesis by down-regulating vascular endothelial growth factor C expression in gastric cancers.

Vascular Endothelial Growth Factor C (VEGF-C) has critical roles in angiogenesis in human cancers; however, the underlying mechanisms regulating VEGF-C expression remain largely unknown. In the present study, VEGF-C protein expression and the density of blood vessels or lymphatic vessels were determined by immunohistochemistry in 103 cases of gastric cancer tissues. Suppression of VEGF-C by miR-27b, miR-101 and miR-128 was investigated by luciferase assays, Western blot and ELISA. The miRNAs expression levels were detected in human gastric cancers by real-time quantitative PCR. Cell proliferation, migration and invasion assays were performed to assess the effect of miRNAs on gastric cancer cells and human umbilical vascular endothelial cells (HUVECs). Our data showed that high VEGF-C expression was significantly associated with increased tumor size, advanced TNM classification and clinical stage, higher microvessel density (MVD) and lymphatic density (LVD), as well as poor survival in patients with gastric cancer. Furthermore, VEGF-C was found to be a direct target gene of miR-27b, miR-101, and miR-128. The expression levels of the three miRNAs were inversely correlated with MVD. Overexpression of miR-27b, miR-101, or miR-128 suppressed migration, proliferation activity, and tube formation in HUVECs by repressing VEGF-C secretion in gastric cancer cells. We conclude that miR-27b, miR-101 and miR-128 inhibit angiogenesis by down-regulating VEGF-C expression in gastric cancers.

In vivo and in vitro effect of hepatocarcinoma lymph node metastasis by upregulation of Annexin A7 and relevant mechanisms.

We unveiled the association of Annexin A7 with vascular endothelial growth factor-C (VEGF-C) and the effect of upregulation of Annexin A7 in Hca-F and Hca-P cells on inhibiting hepatocarcinoma (HCC) lymph node metastasis (LNM) in vitro and in vivo. A total of 200 inbred 615 mice were randomly divided into four equal groups inoculated with Hca-F, Hca-P, FAnxa7-upregulated, and PAnxa7-upregulated cells, respectively. The primary tumor, popliteal, inguinal, and iliac lymph nodes were prepared for immunohistochemical (IHC) staining, real-time quantitative polymerase chain reaction (qRT-PCR) analysis, Western blot, and hematoxylin-eosin (H&E) staining. There was over 50% increase both in the number of FAnxa7-upregulated and PAnxa7-upregulated cells migrated through the filter compared to their controls (FAnxa7-control, Hca-F and PAnxa7-control, Hca-P). However, no significant differences were noted in invasion ability between them (all P > 0.05). Tumor lymph vessels were significantly reduced in FAnxa7-upregulated and PAnxa7-upregulated tumors when compared with Hca-F and Hca-P tumors (all P < 0.05). Blood vessel density did not differ significantly between FAnxa7-upregulated and PAnxa7-upregulated tumors and Hca-F and Hca-P tumors. Enzyme-linked immunosorbent assay (ELISA) for VEGF-C showed that upregulating Annexin A7 decreased VEGF-C secretion in FAnxa7-upregulated and PAnxa7-upregulated cells (P < 0.05). The IHC staining result showed that the level of serum Annexin A7 was found to be statistically higher in all experimental groups than that in the control group (P < 0.05). The present results indicated that alterations in serum Annexin A7 expression may be of prognostic relevance in HCC lymphatic metastasis.

Overcoming inefficient secretion of recombinant VEGF-C in baculovirus expression vector system by simple purification of the protein from cell lysate

The first reports about successfully expressed recombinant proteins with the use of a baculovirus vector were published over 30years ago. Despite the long time of refining this expression system, early problems with the production of baculovirus-derived secretory proteins are still not satisfactorily solved. The high expression level driven by baculoviral promoters often does not result in the desired yield of secreted recombinant proteins, which frequently accumulate inside insect cells and are only partially processed. During our attempts to produce vascular endothelial growth factor C (VEGF-C) with the use of a baculovirus vector we also faced an inefficient secretion of the recombinant protein to culture medium. We were not able to improve the outcome and obtain an acceptable concentration of VEGF-C in the medium by changing the culture conditions or utilizing different signal peptides. However, as a significant amount of native VEGF-C was detected inside the baculovirus-infected cells, we developed a simple method to purify recombinant, glycosylated VEGF-C from a lysate of the cells. The presented results indicate that the lack of a secretory protein in the insect cell culture medium after baculovirus infection does not necessarily signify failure in the production of the protein. As demonstrated by us and contrary to generally accepted views, the lysate of baculovirus-infected cells may constitute a valuable source of the biologically active, secretory protein.

The role of CCL21/CCR7 chemokine axis in breast cancer-induced lymphangiogenesis.

BackgroundTumor-induced lymphangiogenesis facilitates breast cancer progression by generating new lymphatic vessels that serve as conduits for tumor dissemination to lymph nodes and beyond. Given the recent evidence suggesting the implication of C-C chemokine ligand 21/chemokine receptor 7 (CCL21/CCR7) in lymph node metastasis, the aim of our study was to define the role of this chemokine pair in breast cancer-associated lymphangiogenesis.MethodsThe expression analysis of CCL21/CCR7 pair and lymphatic endothelial cell (LEC) markers in breast cancer specimens was performed by means of quantitative real-time PCR. By utilizing CCR7 and CCL21 gene manipulated breast cancer cell implants into orthotopic sites of nude mice, lymphatic vessel formation was assessed through quantitative real-time PCR, immunohistochemistry and immunofluorescence assays. Finally, the lymphangiogenic potential of CCL21/CCR7 was assessed in vitro with primary LECs through separate functional assays, each attempting to mimic different stages of the lymphangiogenic process.ResultsWe found that CCR7 mRNA expression in human breast cancer tissues positively correlates with the expression of lymphatic endothelial markers LYVE-1, podoplanin, Prox-1, and vascular endothelial growth factor-C (VEGF-C). We demonstrated that the expression of CCL21/CCR7 by breast cancer cells has the ability to promote tumor-induced lymph-vascular recruitment in vivo. In vitro, CCL21/CCR7 chemokine axis regulates the expression and secretion of lymphangiogenic factor VEGF-C and thereby promotes proliferation, migration, as well as tube formation of the primary human LECs. Finally, we showed that protein kinase B (AKT) signaling pathway is the intracellular mechanism of CCR7-mediated VEGF-C secretion by human breast cancer cells.ConclusionsThese results reveal that CCR7 and VEGF-C display a significant crosstalk and suggest a novel role of the CCL21/CCR7 chemokine axis in the promotion of breast cancer-induced lymphangiogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0306-4) contains supplementary material, which is available to authorized users.

Expression of COX-2 and VEGF-C in cholangiocarcinomas at different clinical and pathological stages.

The aim of this study was to investigate the expression and clinical significance of cyclooxygenase 2 (COX-2) and vascular en-dothelial growth factor C (VEGF-C) in cholangiocarcinomas at differ-ent clinical and pathological stages. Eighty cholangiocarcinoma sam-ples of patients treated with surgery between January 2012 and January 2014 were collected. Immunohistochemistry was used to detect COX-2 and VEGF-C expression at different clinical and pathological stages. ELISA, real-time PCR, invasive chambers, and MTT assay were ap-plied in cultured cholangiocarcinoma cells treated with a COX-2 inhib-itor. Expression of COX-2 and VEGF-C correlated positively with the clinical TNM stage but did not correlate with the differentiation status. Inhibition of COX-2 activity reduced VEGF-C mRNA expression and secretion in cholangiocarcinoma cells and decreased their migration but not proliferation. Because of its ability to inhibit invasion, COX-2 could be a new target for treatment of cholangiocarcinoma.

Smad4 acts as tumor suppressor by antagonizing lymphangiogenesis in colorectal cancer

Vascular endothelial growth factor-C (VEGF-C) is considered a vital promoter to tumor lymphangiogenesis. Smad4 is an important downstream mediator of transforming growth factor beta (TGF-β) signaling pathway. Smad4 is regarded as a tumor suppressor in colon cancer, but its role in lymphangiogenesis in colorectal cancer is still unclear. In the present study, we investigated the potential inhibiting effect of Smad4 in lymphangiogenesis, and its correlation with VEGF-C secretion in colon cancer. We investigated endogenous Smad4 expression in 7 human colorectal cancer cell lines, and found that six of colon cancer cell lines existed with loss of Smad4. Expression of Smad4 in SW480 cell line is extremely low. SW480 cells were used to establish a Smad4 over-expression model. We investigated the relationship between Smad4 and VEGF-C expression in SW480 cells and found that Smad4 can inhibit the expression of VEGF-C. In an in vitro assay, we found that overexpression of Smad4 can reduce migration and invasion ability in SW480 cells, but had no effect on cell proliferation. Interestingly, Smad4 definitely inhibited the growth in an in vivo assay, and the volumes of transplanted tumors which originated from Smad4 overexpressing SW480 cells in nude mice were smaller than those of normal SW480 cells. In addition, we also demonstrated that Smad4 can inhibit lymphangiogenesis in vivo by immunohistochemistry. In summary, our findings provide molecular evidence that Smad4 may reduce lymphangiogenesis of colon cancer cell by attenuating VEGF-C secretion and act as tumor suppressor by inhibiting migration, invasion and tumorigenicity.

Radiation-induced VEGF-C expression and endothelial cell proliferation in lung cancer.

BackgroundThe present study was undertaken to investigate whether radiation induces the expression of vascular endothelial growth factor C (VEGF-C) through activation of the PI3K/Akt/mTOR pathway,subsequently affecting endothelial cells.Materials and methodsRadiotherapy-induced tumor micro-lymphatic vessel density (MLVD) was determined in a lung cancer xenograft model established in SCID mice. The protein expression and phosphorylation of members of the PI3K/Akt/mTOR pathway and VEGF-C secretion and mRNA expression in irradiated lung cancer cells were assessed by Western blot analysis, enzyme-linked immunosorbent assays (ELISAs), and reverse transcriptase–polymerase chain reaction (RT-PCR). Moreover, specific chemical inhibitors were used to evaluate the role of the PI3K/Akt/mTOR signaling pathway. Conditioned medium (CM) from irradiated control-siRNA or VEGF-C-siRNA-expressing A549 cells was used to evaluate the proliferation of endothelial cells by the MTT assay.ResultsRadiation increased VEGF-C expression in a dose-dependent manner over time at the protein but not at the mRNA level. Radiation also up-regulated the phosphorylation of Akt, mTOR, 4EBP, and eIF4E, but not of p70S6K. Radiation-induced VEGF-C expression was down-regulated by LY294002 and rapamycin (both p < 0.05). Furthermore, CM from irradiated A549 cells enhanced human umbilical vein endothelial cell (HUVEC) and lymphatic endothelial cell (LEC) proliferation, which was not observed with CM from irradiated VEGF-C-siRNA-expressing A549 cells.ConclusionsRadiation-induced activation of the PI3K/Akt/mTOR signaling pathway increases VEGF-C expression in lung cancer cells, thereby promoting endothelial cell proliferation.

KAI1 inhibits lymphangiogenesis and lymphatic metastasis of pancreatic cancer in vivo

Several studies have shown that KAI1 inhibits tumor metastasis, but its mechanism is not clear. The present study aimed to determine the role of KAI1 in lymphatic metastasis, specifically in pancreatic cancer. The KAI1 gene was transfected into the pancreatic cancer cell line MIA PaCa-2 and PANC-1 by using liposomes and selected by G418, and the protein was measured by Western blotting. After successful infection, the cell growth curve was studied by MTT, vascular endothelial growth factor C (VEGF-C) secretion by pancreatic cancer cell were measured by ELISA. The KAI1 and pCMV transfected MIA PaCa-2 cells were renamed as MIA PaCa-2-K and MIA PaCa-2-p. These two kinds of cells were injected into the subcuticular layer of nude mice; both tumor growth and metastasis through the lymphatic nodes were assessed. Lymphangiogenesis in tumors was measured by immunohistochemistry. The VEGF-C secretion was significantly reduced in MIA PaCa-2 cells compared with PANC-1 cells after being transfected with the KAI1 gene. The growth rate of subcutaneous tumors was similar after the injection of MIA PaCa-2-K, MIA PaCa-2, and MIA PaCa-2-p. MIA PaCa-2-K tumors showed slower lymphangiogenesis and lymph node metastasis compared with MIA PaCa-2 and MIA PaCa-2-p tumors. The overexpression of KAI1 inhibits the lymphangiogenesis and lymph node metastasis of MIA PaCa-2 pancreatic tumors.

Lymphatic vessel function and lymphatic growth factor secretion after microvascular lymph node transfer in lymphedema patients.

Background:Recent reports have shown that microvascular lymph node transfer may improve lymphatic drainage in lymphedema patients. Lymphatic anastomoses are expected to form spontaneously in response to lymphatic growth factor [vascular endothelial growth factor C (VEGF-C)] secreted by the transferred lymph nodes.Methods:We have analyzed the results of 19 lymph node transfer patients operated on 2007–2012. Postoperat ive lymphatic function of the affected arm was evaluated using semiquantitative lymphoscintigraphy (transport index) and limb circumference measurements. To investigate the postoperative VEGF-C secretion, we examined axillary seroma fluid samples after different surgical operations, including lymph node transfer.Results:The transport index was improved postoperatively in 7 of 19 patients. Ten of the 19 patients were able to reduce or even discontinue using compression garments. Arm circumferences were reduced in 12 of 19 patients. Six of the 7 patients with preoperative erysipelas infections have not had infectious episodes postoperatively during 15–67 months follow-up. Neuropathic pain was relieved in 5 of 5 patients. VEGF-C protein was detected in the axillary seroma fluid both after lymph node transfer and normal breast reconstruction.Conclusions:Reconstructing the lymphatic anatomy of the axilla with a lymph node flap may offer possibilities that other reconstructive options are lacking. However, we will need further reports and comparative studies about the clinical efficacy of this new promising technique. In addition to the transferred lymph nodes, lymphatic growth factor production may also be induced by other factors related to microvascular breast reconstruction.

Inhibition of lymphangiogenic factor VEGF-C expression and production by the histone deacetylase inhibitor suberoylanilide hydroxamic acid in breast cancer cells

Suberoylanilide hydroxamic acid (SAHA), a potent histone deacetylase (HDAC) inhibitor, has been shown to exert anticancer effects in various types of human cancer and is now used in the clinic for cancer treatment. In addition to cytostatic and cytotoxic activities, SAHA also represses angiogenesis to inhibit tumor growth. However, the effect of SAHA on tumor lymphangiogenesis, a step in which cancer cells produce pro-lymphangiogenic factors such as vascular endothelial growth factor-C (VEGF-C) to stimulate proliferation and migration of lymphatic endothelial cells, remains largely unclear. In this study, we investigated the expression of VEGF-C in breast cancer cell lines and found that VEGF-C was highly expressed in MDA-MB-231, MCF-7, MDA-MB-453 and BT-474 cells. SAHA inhibited VEGF-C expression in a dose-dependent manner in these cell lines. The secretion of VEGF-C into conditioned medium was also suppressed. We cloned human VEGF-C gene promoter and demonstrated that SAHA directly repressed promoter activity in MDA-MB-231 cells. Promoter deletion assay suggested that SAHA repressed VEGF-C via the -185/+38 region which contained several transcription factor binding sites. Notably, we found that SAHA reduced Sp1, but not Sp3 and NF-κB protein levels. Treatment with Sp1 inhibitor mithramycinA also inhibited VEGF-C expression in breast cancer cells. In addition, enforced expression of Sp1 partially rescued the inhibition of VEGF-C by SAHA. Collectively, our results suggest that SAHA inhibits VEGF-C expression in breast cancer cells via transcriptional repression and this drug may exert anti-lymphangiogenic activity in cancer treatment.

Periostin Directly and Indirectly Promotes Tumor Lymphangiogenesis of Head and Neck Cancer

BackgroundMetastasis to regional lymph nodes via lymphatic vessels plays a key role in cancer progression. Tumor lymphangiogenesis is known to promote lymphatic metastasis, and vascular endothelial growth factor C (VEGF-C) is a critical activator of tumor lymphangiogenesis during the process of metastasis. We previously identified periostin as an invasion- and angiogenesis-promoting factor in head and neck squamous cell carcinoma (HNSCC). In this study, we discovered a novel role for periostin in tumor lymphangiogenesis.Methods and FindingsPeriostin overexpression upregulated VEGF-C mRNA expression in HNSCC cells. By using conditioned media from periostin-overexpressing HNSCC cells, we examined tube formation of lymphatic endothelial cells. Conditioned media from periostin-overexpressing cells promoted tube formation. To know the correlation between periostin and VEGF-C, we compared Periostin expression with VEGF-C expression in 54 HNSCC cases by immunohistochemistry. Periostin expression was correlated well with VEGF-C expression in HNSCC cases. Moreover, correlation between periostin and VEGF-C secretion was observed in serum from HNSCC patients. Interestingly, periostin itself promoted tube formation of lymphatic endothelial cells independently of VEGF-C. Periostin-promoted lymphangiogenesis was mediated by Src and Akt activity. Indeed possible correlation between periostin and lymphatic status in periostin-overexpressing xenograft tumors and HNSCC cases was observed.ConclusionsOur findings suggest that periostin itself as well as periostin-induced upregulation of VEGF-C may promote lymphangiogenesis. We suggest that periostin may be a marker for prediction of malignant behaviors in HNSCC and a potential target for future therapeutic intervention to obstruct tumoral lymphatic invasion and lymphangiogenesis in HNSCC patients.