Tumor-Derived Exosomes Promote Tumor Growth Through Modulating Microvascular Hemodynamics in a Human Ovarian Cancer Xenograft Model.

OBJECTIVE: Abnormal tumor vascular network contributes to aberrant blood perfusion and reduced oxygenation in tumors, which lead to poor efficacy of chemotherapy and radiotherapy. We aimed to explore the effects of the tumor-derived exosomes (TDEs) and C188-9 (a small molecule inhibitor of signal transducer and activator of transcription 3, STAT3) on tumor microvascular hemodynamics and determine which blood flow oscillations for various frequency intervals are responsible for these changes. METHODS: Microvascular hemodynamics parameters were recorded using a PeriFlux 6000 EPOS system in tumor surface in a nude mouse subcutaneous xenograft model. Oscillations of laser Doppler flowmetry (LDF) signal were investigated by wavelet transform analysis. RESULTS: TDEs facilitated tumor growth at least partially was associated with increasing blood flow in smaller vessels with lower speed and decreasing the blood flow at larger vessels with higher speed. Lower oxyhemoglobin saturation (SO2) on tumor surface was aggravated by TDEs, and C188-9 treatment significantly alleviated this decrease. Wavelet transform spectral analysis revealed that TDEs increased the amplitude of oscillations in four frequency intervals related to endothelial (NO-dependent and -independent), myogenic and neurogenic activities, and C188-9 had no effect on this increase. CONCLUSIONS: TDEs facilitated tumor growth partially was associated with increasing blood flow in distributing vessels, reducing blood perfusion in larger vessels, and lowering SO2 on tumor surface. Enhanced vascular smooth muscle, endothelial and neurogenic activities occurred in tumor superficial zone.

Sodium Pentobarbital Suppresses Breast Cancer Cell Growth Partly via Normalizing Microcirculatory Hemodynamics and Oxygenation in Tumors.

Breast cancer remains the leading cause of cancer-related death among women worldwide. Sodium pentobarbital was found to play an inhibitory role in glioma growth in rats. In this study, we aimed to evaluate the effects of sodium pentobarbital on breast cancer growth both in vitro and in vivo, and its impacts on the microcirculatory changes on both skin and tumor surface in mice bearing subcutaneous xenograft. Cell counting assay was used to assess the antiproliferative effect of sodium pentobarbital on MDA-MB-231 breast cancer cells. Subcutaneous xenograft model was established to study the role of sodium pentobarbital on in vivo tumor growth. Speed-resolved blood perfusion, hemoglobin oxygen saturation (SO2, %), total hemoglobin tissue concentration (ctTHb, µM), and red blood cell (RBC) tissue fraction (%) were examined simultaneously by using enhanced perfusion and oxygen saturation system to investigate the effects of sodium pentobarbital on microcirculatory hemodynamics and oxygenation. Sodium pentobarbital suppressed breast tumor growth both in vitro and in vivo. Cutaneous blood flux in nutritive capillaries with low-speed flow was significantly increased in tumor-bearing mice, and high-dose sodium pentobarbital treatment cause a reduction in this low-speed blood flux, whereas sodium pentobarbital therapy caused an elevated blood flux in larger microvessels with mid and high speed in a dose-dependent manner. Different doses of sodium pentobarbital exerted different actions on SO2, ctTHb, and RBC tissue fraction. Collectively, the inhibitory effect of sodium pentobarbital on breast tumor growth was at least partly associated with its ability to normalize microcirculatory hemodynamics and oxygenation in tumors. SIGNIFICANCE STATEMENT: This study is the first to demonstrate the inhibiting effect of sodium pentobarbital on breast cancer growth both in vitro and in vivo, and such an inhibition was at least partly associated with its ability to normalize microcirculatory hemodynamics and oxygenation in tumors.

Roquin2 suppresses breast cancer progression by inhibiting tumor angiogenesis via selectively destabilizing proangiogenic factors mRNA.

Tumor angiogenesis is an essential step in tumor growth and metastasis. The initiation of tumor angiogenesis is dictated by a shift in the balance between proangiogenic and antiangiogenic gene expression programs. Roquin2 is a zinc-finger RNA-binding protein with important roles in mediating the expression of inflammatory genes, such as TNF, IL6 and PTGS2, which are also important angiogenic factors. In this study, we demonstrate that Roquin2 functions as a potent tumor angiogenesis regulator that inhibits breast tumor-induced angiogenesis by selectively destabilizing mRNA of proangiogenic gene transcripts, including endoglin (ENG), endothelin-1 (EDN1), vascular endothelial growth factor B (VEGFB) and platelet derived growth factor C (PDGFC). Roquin2 recognizes and binds the stem-loop structure in the 3'untranslated region (3'UTR) of these mRNAs via its ROQ domain to destabilize mRNA. Moreover, we found that Roquin2 expression was reduced in breast cancer cells and tissues, and associated with poor prognosis in breast cancer patients. Overexpression of Roquin2 inhibited breast tumor-induced angiogenesis in vitro and in vivo, whereas silencing Roquin2 enhanced tumor angiogenesis. In vivo induction of Roquin2 by adenovirus significantly suppressed breast tumor growth, metastasis and angiogenesis. Taken together, our results identify that Roquin2 is a novel breast cancer suppressor that inhibits tumor angiogenesis by selectively downregulating the expression of proangiogenic genes.

RNA-binding protein SAMD4A inhibits breast tumor angiogenesis by modulating the balance of angiogenesis program.

Tumor-induced angiogenesis is important for further progression of solid tumors. The initiation of tumor angiogenesis is dictated by a shift in the balance between proangiogenic and antiangiogenic gene expression programs. However, the potential mechanism controlling the expression of angiogenesis-related genes in the tumor cells, especially the process mediated by RNA-binding protein (RBP) remains unclear. SAMD4A is a conserved RBP across fly to mammals, and is believed to play an important role in controlling gene translation and stability. In this study, we identified the potential role of SAMD4A in modulating angiogenesis-related gene expression and tumor progression in breast cancer. SAMD4A expression was repressed in breast cancer tissues and cells and low SAMD4A expression in human breast tumor samples was strongly associated with poor survival of patients. Overexpression of SAMD4A inhibited breast tumor angiogenesis and caner progression, whereas knockdown of SAMD4A demonstrated a reversed effect. Mechanistically, SAMD4A was found to specifically destabilize the proangiogenic gene transcripts, including C-X-C motif chemokine ligand 5 (CXCL5), endoglin (ENG), interleukin 1ß (IL1ß), and angiopoietin 1 (ANGPT1), by directly interacting with the stem-loop structure in the 3' untranslated region (3'UTR) of these mRNAs through its sterile alpha motif (SAM) domain, resulting in the imbalance of angiogenic genes expression. Collectively, our results suggest that SAMD4A is a novel breast tumor suppressor that inhibits tumor angiogenesis by specifically downregulating the expression of proangiogenic genes, which might be a potential antiangiogenic target for breast cancer therapy.

Ovarian cancer derived PKR1 positive exosomes promote angiogenesis by promoting migration and tube formation in vitro.

Cancer cell derived exosomes play important roles in cancer progression and modulation of the tumour microenvironment. This study aims to investigate the role of prokineticin receptor 1 (PKR1) positive exosomes on angiogenesis. In the present study, PKR1 expression in tumour samples from ovarian cancer patients were examined firstly. Then, two ovarian cancer cell lines, namely A2780 and HO-8910 cells, were used to isolate and obtain the PKR1 positive exosomes from the serum free medium. The function analysis of PKR1 positive exosomes on angiogenesis was conducted by cell proliferation and migration assay, tube formation analysis, and tumour volume assay. The results showed that PKR1 expression was down regulated in tumour samples of ovarian cancer patients compared with adjacent normal tissues. The intracellular expression of PKR1 could be detected in A2780 and HO-8910 cells. And, the isolated exosomes from the serum free medium were confirmed by transmission electron microscopic and NTA analysis, as well as the co-presence of PKR1 with exosome marker CD63. The function analysis of PKR1 positive exosomes on angiogenesis demonstrated the uptake of PKR1 positive exosomes by human umbilical vein endothelial cells through immunofluorescence staining. The angiogenesis assays in vitro indicated that PKR1 positive exosomes promoted migration and tube formation of HUVECs but not proliferation. The endogenous PKR1 was also verified to help to enhance migration and promote tube formation of vascular endothelial cells, which might involved in the phosphorylation of STAT3. Additionally, The tumour volume from exosomes treated A2780 tumour-bearing mice was significantly increased compared with the control group, accompanied with the induced PKR1 expression and phosphorylation of STAT3 level. SIGNIFICANCE OF THE STUDY: This study proved the important role of PKR1 positive exosomes released from ovarian cancer cells on promoting angiogenesis. The data indicated that PKR1 derived from ovarian cancer cells could act as an important tumour associated antigen and biomolecular factor for cellular communication in tumour microenvironment.

The circulating level of miR-122 is a potential risk factor for endothelial dysfunction in young patients with essential hypertension.

MicroRNAs are key molecules involved in the regulation of endothelial function. They are important risk factors and biomarkers for the development of hypertension related to endothelial dysfunction. However, the gene expression patterns associated with hypertension development related to endothelial dysfunction have not been fully elucidated. We conducted a case-control study of 65 patients with essential hypertension (EH) and 61 controls without EH. Plasma levels of miR-122 and its target protein high-affinity cationic amino acid transporter 1 (CAT-1) were measured by qRT-PCR and ELISA, respectively. miR-122 expression in plasma of patients with EH was significantly higher than that of the control group (p = 0.001), while CAT-1 expression in patients with EH was significantly lower than that in the control group (p = 0.018). miR-122 expression in plasma of young patients with EH was significantly higher than that in young people without EH (p = 0.0004), and CAT-1 expression in plasma of young patients with EH was also significantly lower than that of the control group (p = 0.002). CAT-1 expression in the plasma of young participants was significantly higher than that of individuals aged ≥40 years (p = 0.003), whereas miR-122 expression was significantly lower (p = 0.001). We showed that among patients with EH, the high expression of miR-122 contributed to endothelial dysfunction by suppressing the expression of the CAT-1 protein, which led to a decrease in CAT-1 expression in plasma. Therefore, high expression of miR-122 appears to be a risk factor for endothelial dysfunction in EH, especially in younger patients.

The anti-angiogenic effect of dexamethasone in a murine hepatocellular carcinoma model by augmentation of gluconeogenesis pathway in malignant cells.

PURPOSE: Angiogenesis is a long-term complex process involving various protein factors in hepatocellular carcinoma (HCC). Dexamethasone (Dex), considered as a synthetic glucocorticoid drug in clinical therapy, has been reported to have the therapeutic efficacy against liver cancer by intervention of abnormal glycolysis. In this study, we investigated the anti-angiogenic effect of Dex in murine liver cancer and attempted to demonstrate the potential mechanism. METHODS: The malignant cells H22 were treated with Dex. Western blotting was used to explore the expression of PEPCK and G6Pase which were the two key enzymes that regulated gluconeogenesis. The supernatants from cultured H22 treated by Dex were collected and co-cultured with HUVECs. In vitro, migration assay, transwell assay and tube formation assay were performed to assess for migration, proliferation and tube formation abilities of HUVECs, respectively. In situ murine hepatoma model with green fluorescent protein markers (HepG2-GFP) was constructed to determine angiogenesis after treatment by Dex. RESULTS: PEPCK and G6Pase were almost deficient in H22 compared with normal liver cells NCTC-1469 (P < 0.01). After treated by Dex, the gluconeogenesis could be restored significantly (P < 0.01) in H22 cells. The supernatant of H22 treated by Dex inhibited the migration, tube formation and endothelial permeability in HUVECs (P < 0.05). In mouse tissue, PEPCK and G6Pase were highly expressed in Dex group than control groups (P < 0.01). 11ß-HSDs abnormally expressed in tumor also could be restored by Dex. Meanwhile, the density and total length of microvessels in Dex-treated group were less than those in HCC groups (P < 0.05). CONCLUSIONS: This study explored the therapeutic efficacy of Dex in murine HCC. Dex might inhibit tumor growth and angiogenesis by augmenting the gluconeogenesis pathway.

Decreased expression of MDR1 in PEG-conjugated hemoglobin solution combined cisplatin treatment in a tumor xenograft model.

This study aims to examine the contribution of PEG-conjugated hemoglobin combined with cisplatin to the expression of HIF-1α and MDR1 in a tumor xenograft model. Cervical carcinoma models were assigned to 4 groups and treated respectively: group 1(control); group 2, cisplatin; group 3, PEG-Hb; group 4 cisplatin plus PEG-Hb. 4 weeks later, tumor volume and MVD was significantly decreased in group 4 compared with other groups. Lower expression of HIF-1α and MDR1 were detected in group4. Taken together, our data indicated that PEG-Hb plus cisplatin can promote tumor tissue oxygenation and enhance the chemotherapy sensitivity. HIF-1α regulated MDR1 pathway correlated with this process.

PEG-conjugated hemoglobin combination with cisplatin enforced the antiangiogeic effect in a cervical tumor xenograft model.

Angiogenesis, an essential event involved in a tumor's progression and metastasis, is regulated by hypoxia. Hypoxia widely exists in solid tumors due to the abnormal vasculature of tumor tissue and insufficiency of tissue oxygenation. We speculate that hemoglobin-based oxygen carriers (HBOCs) can attenuate tissue hypoxia, thereby suppressing the angiogenesis in solid tumor in the context that HBOCs have the ability to increase tissue oxygenation. In the present study, PEG-conjugated hemoglobin solution (0.3 g/kg i.v. or 0.6 g/kg i.v.) was intravenously administrated to BALB/c nude mice bearing the cervical tumor twice a week with or without the treatment of cisplatin (5mg/kg i.p.) to investigate whether PEG-conjugated hemoglobin has a chemo-sensitization effect though anti-angiogenesis pathway. Tumor volume was measured every three days and tumor hypoxia was detected by immunohistochemistry for Hypoxyprobe-1. Anti-angiogenic effect was accessed by detection of mRNA and protein levels of vascular endothelial growth factor (VEGF), the most important angiogenic factor. Our results showed that high concentration of PEG-conjugated hemoglobin solution significantly impeded the growth of tumor when compared with the control group. Moreover, VEGF expression was declined when treated with PEG-conjugated hemoglobin, possibly through the HIF regulation system. Collectively, treatment of PEG-conjugated hemoglobin combination with cisplatin has an antiangiogenic effect, but the underlying mechanism should be further studied.

Influence of PEG-conjugated hemoglobin on tumor oxygenation and response to chemotherapy.

Hypoxic tumors are significantly more malignant, metastatic, radio- and chemoresistant. The use of artificial oxygen carriers represents a new approach to the problem of hypoxia. In the present study, female athymic BALB/c nude mice bearing the cervical carcinoma were untreated or treated with cisplatin to determine whether administration of artificial oxygen carrier (PEG-conjugated Hemoglobin, PEG-Hb) could improve the tumor oxygenation and enhance the anti-tumor efficacy of cisplatin. Pimonidazole staining was employed to detect tumor tissue oxygenation status. We found that the application of a higher dose (0.6 g/kg) PEG-Hb could significantly ameliorate the hypoxic condition in cervical carcinoma xenograft models. Co-administration of PEG-Hb (0.6 g/kg) with cisplatin produced significant tumor growth inhibition and pro-apoptotic and anti-proliferative effects as compared to cisplatin alone. These suggest the evaluated PEG-Hb in this experiment has positive effects on cisplatin or cisplatin-based chemotherapy, and further work to optimize its application is warranted.

Effect of artificial oxygen carrier with chemotherapy on tumor hypoxia and neovascularization.

UNLABELLED: In addition to transfusion alternatives, artificial oxygen carriers are a benefit in ischemia disorders. This study aimed at evaluating the possible effects of PEG-conjugated hemoglobin (PEG-Hb) plus cisplatin on tumor hypoxia and neovasculature. METHODS: HeLa cells were injected into submucosa of golden hamster cheek pouch to build tumor model. Animals were randomly assigned to 4 groups (n=10) and treated respectively: group 1, saline; group 2, cisplatin (5mg/kg); group 3, cisplatin (5mg/kg) plus PEG-Hb (0.3g/kg); group 4, cisplatin (5mg/kg) plus PEG-Hb (0.6g/kg). Tumor neovascularization morphological variation and tissue hypoxia were detected by intravital microscopy and immunostaining, respectively. RESULTS: Microvessel tortuosity and area capillary density in peritumoral areas were notably depressed in group 4 compared with group 2 (p<0.05). Hypoxia markers pimonidazole and HIF-1alpha expression were decreased significantly in group 4. CONCLUSION: PEG-Hb in high concentration can notably improve tumor tissue oxygenation and normalize neovasculature; it may be a potential adjuvant to chemotherapy in cancer.

Cisplatin up-regulates ICAM-1 expression in endothelial cell via a NF-kappaB dependent pathway.

The anticancer drug cis-diammindichloroplatin (cisplatin) can cause severe side-effects, but to date, the mechanisms of action of these dangerous side-effects have not been completely elucidated. Since cellular adhesion molecules (CAMs), by mediating the recruitment of circulating leukocytes to the blood vessel wall and their subsequent migration into the subendothelial spaces, play a crucial role in several pathophysiologic processes, we sought potential proof for CAMs in the pathophysiology of cisplatin-induced vascular damage. In vitro, human umbilical vein endothelial cells (HUVECs) were subjected to various concentrations of cisplatin, considerable up-regulation of intercellular adhesion molecule-1 (ICAM-1) but not P-selectin, E-selectin, and vascular cell adhesion molecule 1 at both messenger mRNA and protein expression levels were observed. Electrophoretic mobility shift assays and Western blotting analysis revealed that cisplatin up-regulates ICAM-1 expression in HUVECs via an NF-kappaB-dependent pathway. Further intravital microscopy study demonstrated that significantly higher (P < 0.01) numbers of rolling and sticking leukocytes on the wall of postcapillary venules in male Golden Syrian hamster's cheek pouch bearing a human cervical carcinoma were observed, while inhibition of ICAM-1 by using specific anti-ICAM-1 antibody can attenuate cisplatin-stimulated leukocyte/endothelium interactions. These data suggest that ICAM-1 involves in the pathophisiologic process of cisplatin-induced vascular toxicity and may be exploited for therapeutic advantage.

Melatonin modulates the expression of VEGF and HIF-1 alpha induced by CoCl2 in cultured cancer cells.

Melatonin is an important natural oncostatic agent. At present there are no data available as to its possible influence on tumor angiogenesis, which is a major biological mechanism responsible for tumor growth and dissemination. It is well known that vascular endothelial growth factor (VEGF) is crucial to a solid tumor's higher vascularization and development. To investigate the possible influence of melatonin on angiogenesis, we studied the effect of melatonin on endogenous VEGF expression in three human cancer cell lines (PANC-1, HeLa and A549 cells). In this study, we report that physiologic concentrations of melatonin have no obvious impact on the VEGF expression, whereas pharmacologic concentrations of melatonin suppress the VEGF mRNA and protein levels induced by hypoxia mimetic cobalt chloride (CoCl(2)). Melatonin also decreases hypoxia-inducible factor (HIF)-1alpha protein levels, suggesting a role for transcription factor HIF-1 in the suppression of VEGF expression. The effect of pharmacologic concentrations of melatonin on VEGF and HIF-1alpha under normoxia is uncertain, which indicates that the regulatory mechanisms of VEGF in the absence or presence of CoCl(2) are different and other or additional transcription factors may be involved. Taken together, our data show that melatonin in high concentrations markedly reduces the expression of endogenous VEGF and HIF-1alpha induced by CoCl(2) in cultured cancer cells.

Intracellular signaling pathways involved in cell growth inhibition of human umbilical vein endothelial cells by melatonin.

Melatonin, an indolamine mainly produced in the pineal gland, has received a great deal of attention in the last decade because of its oncostatic effects, which are due to its immunomodulatory, antiproliferative, antioxidant and its possible antiangiogenesis properties. Herein, we document its antiproliferative action on human umbilical vein endothelial cells (HUVECs). Moreover, the possible cell signaling pathways when melatonin inhibited HUVEC proliferation were explored in this study. Primary HUVECs were isolated, cultured, purified and identified before the studies were performed. HUVECs were found to possess G-protein-coupled membrane receptors for melatonin (MT1 and MT2) and also nuclear melatonin receptors (RORalpha and RORbeta, especially RORbeta). No obvious expression of RORgamma was found. We investigated the membrane receptors and several intracellular signaling pathways including mitogen-activated protein kinases (MAPK)/extracellular signal-related kinases (ERK), phosphoinositol-3-kinase (PI3K)/Akt and protein kinases C (PKC) involved in antiproliferative action of melatonin on HUVECs. The blockade of these pathways using special inhibitors decreased cell growth. Furthermore, the constitutive activation of nuclear factor kappa B (NF-kappaB) contributed to the proliferation of HUVECs. High concentrations of melatonin inhibited both NF-kappaB expression and its binding ability to DNA, possibly through inactivation of ERK/Akt /PKC pathways. Taken together, high concentrations of melatonin markedly reduced HUVEC proliferation; the antiproliferative action of melatonin was closely correlated with following pathway: melatonin receptors/ERK/PI3K/Akt/PKC/ NF-kappaB.

[Correcting the congenital ala defect of the facial cleft with revolving reposition].

OBJECTIVE: To introduce the experience of correcting the congenital ala defect of facial cleft with the method of revolving reposition. METHODS: Five patients were treated with this method. Based on features of the defect, two different operations were employed with the medial crura or the lateral crura of the ala as the revolving pivot. RESULTS: All the operations obtained good results. CONCLUSION: The operation of revolving reposition for congenital ala defect is a simple practical method with satisfactory results.