Jing Ma

RNAi-mediated silencing of VEGF-C inhibits non-small cell lung cancer progression by simultaneously down-regulating the CXCR4, CCR7, VEGFR-2 and VEGFR-3-dependent axes-induced ERK, p38 and AKT signalling pathways

Vascular endothelial growth factor C (VEGF-C) expression is associated with the malignant tumour phenotype making it an attractive therapeutic target. We investigated the biological roles of VEGF-C in tumour growth, migration, invasion and explored the possibility of VEGF-C as a potential therapeutic target for the treatment of non-small cell lung cancer (NSCLC). A lentivirus-mediated RNA interference (RNAi) technology was used to specifically knockdown the expression of VEGF-C in A549 cells. Quantitative reverse transcriptase-polymerase chain reaction, flow cytometry, Western blot, immunohistochemistry, cellular growth, migration, invasion and ELISA assays were used to characterise VEGF-C expression in vitro. A lung cancer xenograft model in nude mice was established to investigate whether knockdown of VEGF-C reduced tumour growth in vivo. Silencing of VEGF-C suppressed tumour cell growth, migration and invasion in vitro; suppressed tumour growth, angiogenesis and lymphangiogenesis by tail vein injection of lentivirus encoded shRNA against VEGF-C in vivo. More importantly, silencing of VEGF-C also trapped the VEGFR-2, VEGFR-3, CXCR4, CCR7-dependent axes, and down-regulated the AKT, ERK and p38 signalling pathways. These results suggest that VEGF-C has a multifaceted role in NSCLC growth, migration and invasion; that VEGF-C-mediated autocrine loops with their cognate receptors and chemokine receptors are significant factors affecting tumour progression; and that RNAi-mediated silencing of VEGF-C represents a powerful therapeutic approach for controlling NSCLC growth and metastasis.

Roles of VEGF-C and Smad4 in the Lymphangiogenesis, Lymphatic Metastasis, and Prognosis in Colon Cancer

Background/AimsWe combined two different signal pathways on transforming growth factor β1 (TGF-β1)-Smad and vascular endothelial growth factor C (VEGF-C)/VEGF receptors for exploring changes in pathway members and their influence on lymphangiogenesis and clinicopathological features.Materials and MethodsExpression of TGF-β1, TGF-βRII, Smad4, VEGF-C, and VEGFR-3 was immunohistochemically evaluated in 147 colon cancer patients who were followed up for 5xa0years.ResultsLymphatic vessel density in colon cancer tissues was significantly higher than in normal colonic tissues. Smad4 expression negatively correlated with lymphatic vessel count and VEGF-C expression. VEGF-C expression positively correlated with lymphatic vessel count. Analysis using the Kaplan–Meier method indicated that patients with VEGF-C-positive tumors had significantly shorter overall survival and tumor-free survival time than those with VEGF-C-negative tumors. Patients with Smad4-negative tumors had significantly shorter overall survival and tumor-free survival time than those with Smad4-positive tumors.ConclusionsBoth Smad4 and VEGF-C are involved in lymphangiogenesis and lymphatic metastasis. Smad4 and VEGF-C expression may be clinically useful indicators for prognostic evaluation in colon cancer patients.

Expression of VEGF-C and VEGF-D as Significant Markers for Assessment of Lymphangiogenesis and Lymph Node Metastasis in Non-Small Cell Lung Cancer

Vascular endothelial growth factor (VEGF)‐C and VEGF‐D induce lymphangiogenesis through activation of VEGF receptor 3 (VEGFR‐3) and have been implicated in tumor spread to the lymphatic system. Lymph node dissemination critically determines clinical outcome and therapeutic options of patients with non‐small cell lung cancer (NSCLC). However, the relationship of VEGF‐C, VEGF‐D, and lymph node metastasis in cancers, including NSCLC, is still controversial. To evaluate the relationship between lymphangiogenesis and lymph node metastasis, the expression of VEGF‐C and VEGF‐D in NSCLC tumors were detected by immunohistochemistry and quantitative real‐time polymerase chain reaction (QRT‐PCR). QRT‐PCR revealed that in marginal region VEGF‐C and VEGF‐D mRNA was significantly higher than in tumor center, and VEGF‐D mRNA was also higher than that in peritumoral lung tissue. Immunohistochemically, we observed the same heterogeneous expression of VEGF‐C and VEGF‐D proteins. The group with high expression of VEGF‐C and VEGF‐D in marginal region had a higher incidence of lymph node metastasis compared with the group with low expression. Furthermore, the group with high expression of VEGF‐D in marginal region had a higher incidence of lymphatic invasion. The group with high peritumoral lymphatic vessel density (LVD) had higher expression of VEGF‐C and VEGF‐D mRNA compared with the group with low peritumoral LVD. Our studies suggested that the expression of VEGF‐C and VEGF‐D at invasive edge was significantly associated with lymph node metastasis or lymphatic invasion in patients with NSCLC and may be involved in regulation of lymphangiogenesis and lymph node metastasis in NSCLC. Anat Rec, 2010.

Lymphangiogenesis and Its Relationship With Lymphatic Metastasis and Prognosis in Malignant Melanoma

Regional lymph node metastasis is one of the important indicators of cutaneous malignant melanoma. Newly formed lymphatic vessels are considered to provide a route whereby tumor cells can migrate to the lymph nodes. Both vascular endothelial growth factors (VEGF) ‐C and ‐D have been confirmed to participate in tumor lymphangiogenesis, but the prognostic significance of VEGF‐C, VEGF‐D, and lymphangiogenesis in cutaneous malignant melanoma remains controversial. To clarify the effects of these factors and to evaluate the relationships between lymphangiogenesis, lymph node metastasis, and prognosis in patients with malignant melanoma, the expressions of VEGF‐C, VEGF‐D, and their receptor (VEGFR) ‐3 were detected by immunohistochemistry and reverse transcriptase‐polymerase chain reaction. The expressions of both VEGF‐C and VEGF‐D proteins were concomitantly detected in the cytoplasm of the malignant cells. VEGF‐C and VEGF‐D expressions were associated with VEGFR‐3 expression and were significantly correlated with both peritumoral lymphangiogenesis and lymph node metastasis. The incidence of peritumoral lymphatic vessels was significantly higher in lymph node metastatic melanomas than that in nonmetastatic melanomas. Univariate and multivariate analyses indicated that VEGF‐C and VEGF‐D were independent prognostic factors for overall survival and disease‐free survival in patients with malignant melanoma. This study suggests that both VEGF‐C and VEGF‐D are involved in peritumoral lymphangiogenesis and lymphatic metastasis. VEGF‐C and VEGF‐D expression may be clinically useful indicators for prognostic evaluation in patients with cutaneous malignant melanoma. Anat Rec, 2008.

UCF-101, A Novel Omi/HtrA2 Inhibitor, Protects Against Cerebral Ischemia/Reperfusion Injury in Rats

The aim of this study was to investigate the therapeutic efficacy and neuroprotective mechanisms of UCF‐101, a novel Omi/HtrA2 inhibitor, following ischemia/reperfusion brain injury. Male Wistar rats were subjected to 2 hr of middle cerebral artery occlusion followed by reperfusion. Animals were divided into 3 groups: sham, vehicle‐treated ischemia/reperfusion, and UCF‐101 treatment. In the UCF‐101 treatment group, rats were intraperitoneally administered UCF‐101 (1.5 μmol/kg) 10 min prior to reperfusion. The rats were evaluated for neurological deficits, and brain infarct volume was assessed by 2,3,5‐triphenyl tetrazolium chloride. TUNEL staining was utilized to evaluate the amount of apoptosis. In addition, expressions of protein caspase‐8, caspase‐3, FasL, and FLIP were examined by Western blot analysis. Results demonstrated that UCF‐101 treatment significantly decreased cerebral infarct size by about 16.27% (P < 0.05) and also improved neurological behavior. TUNEL staining revealed that UCF‐101 treatment significantly reduced TUNEL‐positive cells in the cerebral cortex. Furthermore, the upregulation in the expression of FasL and the cleavage products of active caspase‐8 and caspase‐3 induced by ischemia was attenuated in mice treated with UCF‐101, whereas upregulation of FLIP levels was increased. The present results demonstrated that UCF‐101 protects against cerebral ischemia/reperfusion injury in mice. UCF‐101 provided neuroprotection in vivo, and this was correlated with regulation of Fas‐mediated apoptotic proteins. Taken together, the use of UCF‐101 is a potent, neuroprotective factor for the treatment of focal cerebral ischemia. Anat Rec, 292:849–856, 2009.

Arctigenin Anti‐Tumor Activity in Bladder Cancer T24 Cell Line Through Induction of Cell‐Cycle Arrest and Apoptosis

Bladder cancer is the most common neoplasm in the urinary system. This study assesses arctigenin anti‐tumor activity in human bladder cancer T24 cells in vitro and the underlying molecular events. The flow cytometry analysis was used to detect cell‐cycle distribution and apoptosis. Western blotting was used to detect changes in protein expression. The data showed that arctigenin treatment reduced viability of bladder cancer T24 cells in a dose‐ and time‐dependent manner after treatment with arctigenin (10, 20, 40, 80, and 100 μmol/L) for 24 hr and 48 hr. Arctigenin treatment clearly arrested tumor cells in the G1 phase of the cell cycle. Apoptosis was detected by hoechst stain and flow cytometry after Annexin‐V‐FITC/PI double staining. Early and late apoptotic cells were accounted for 2.32–7.01% and 3.07–7.35%, respectively. At the molecular level, arctigenin treatment decreased cyclin D1 expression, whereas CDK4 and CDK6 expression levels were unaffected. Moreover, arctigenin selectively altered the phosphorylation of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 and activated phosphorylation of p38 significantly in a dose‐dependent manner. These results suggest that arctigenin may inhibit cell viability and induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen‐activated protein kinase pathway may play an important role in the anti‐tumor effect of arctigenin. The data from the current study demonstrate the usefulness of arctigenin in bladder cancer T24 cells, which should further be evaluated in vivo before translation into clinical trials for the chemoprevention of bladder cancer. Anat Rec, 2012.

Intratumoral Lymphatics and Lymphatic Vessel Invasion Detected by D2-40 Are Essential for Lymph Node Metastasis in Bladder Transitional Cell Carcinoma

Bladder cancer is frequently associated with regional lymph node metastasis at the time of diagnosis or after initial treatment, and lymph node metastasis is crucial for clinical therapeutic strategies. Lymphangiogenesis, detected by antibodies specific for lymphatic endothelial cells, is correlated with cancer spread, but the mechanisms that underlie lymphatic spread and the role of lymphangiogenesis in cancer metastasis has been less clear. The aim of this study was to investigate the association of vascular endothelial growth factor (VEGF)‐D expression, intratumoral lymphatics, and lymphatic invasion associated with lymph node metastasis as well as the prognostic analysis in patients with bladder transitional cell carcinoma (TCC). The VEGF‐D expression was evaluated by immunohistochemistry in 72 specimens, and tumoral lymphatic vessels were measured by D2‐40. Counts of lymph vessels were taken in intratumoral and peritumoral areas. Survival analyses and their independent roles were investigated using univariate and multivariate analysis models. The high expression of VEGF‐D was closely associated with the intratumoral lymphatic vessels, tumoral lymphatic invasion, and lymph node metastasis as well as a shorter overall survival. Higher lymphatic vessel density, intratumoral lymphatics, and lymphatic invasion showed a significant association with lymph node metastasis. Univariate analysis indicated that VEGF‐D, intratumoral lymphatics, and lymphatic invasion were associated with overall survival, but they were not independent prognostic factors for bladder TCC in multivariate analysis. We conclude that VEGF‐D plays an essential role in tumoral lymphangiogenesis. Intratumoral lymphatics and lymphatic invasion are important predictive factors of pelvic lymph node metastasis in patients with bladder cancer. Anat Rec, 2010.

Vascular Endothelial Growth Factor D and Intratumoral Lymphatics as Independent Prognostic Factors in Epithelial Ovarian Carcinoma

Lymph node metastasis is an important prognostic indicator for disease progression and is crucial for therapeutic strategies of epithelial ovarian carcinoma. Vascular endothelial growth factor (VEGF)‐D has been confirmed to have potent lymphangiogenic function in experimental models, but the role in the progression of human ovarian carcinoma remains presently controversial. The purpose of this study was to investigate the prognostic significance of VEGF‐D and the presence of intratumoral lymphatics in patients with epithelial ovarian carcinoma. The VEGF‐D expression was evaluated by immunohistochemistry in 78 specimens of epithelial ovarian carcinoma and tumoral lymphatic vessels were measured by D2‐40. The expression of VEGF‐D protein was detected in the cytoplasm of the tumor cells and in stroma occasionally. The high expression of VEGF‐D was closely associated with the FIGO stage, intratumoral lymphatic vessels, tumoral lymphatic invasion, and lymph node metastasis as well as a shorter overall survival. Univariate and multivariate analysis indicated that VEGF‐D, intratumoral lymphatics, and lymphatic invasion were independent prognostic factors for overall survival and disease‐free survival in patients with epithelial ovarian carcinoma. We conclude that VEGF‐D plays an essential role in tumoral lymphangiogenesis and lymphatic spread, VEGF‐D expression, and the intratumoral lymphatics may be clinically useful indicators for prognostic evaluation in patients with epithelial ovarian carcinoma. Anat Rec, 2009.

Blockade of the intermediate-conductance Ca2+-activated K+ channel inhibits the angiogenesis induced by epidermal growth factor in the treatment of corneal alkali burn

Epidermal growth factor (EGF) is used to treat alkali-burned corneas. However, EGF-induced corneal angiogenesis, which is currently untreatable, is a side effect of this therapy. We therefore explored the role of the intermediate-conductance Ca(2+)-activated K(+) channel (KCa3.1) in EGF-induced angiogenesis and tested whether KCa3.1 blockade can suppress EGF-induced corneal angiogenesis. The proliferation, migration and tube formation of HUVECs (human umbilical vein endothelial cells) in response to EGF, the MEK inhibitor PD98059 and the KCa3.1 inhibitor TRAM-34 were analyzed inxa0vitro via MTT, cell counting, scratch and tube formation assays. The protein and mRNA levels of KCa3.1, phosphorylated-ERK (P-ERK), total-ERK (T-ERK), cyclin-dependent kinase 4 (CDK4), vimentin and MMP-2 were assessed via western blotting and RT-PCR. KCa3.1 and vimentin expression were also detected through immunofluorescence staining. Flow cytometry was performed to examine the cell cycle. Further, an inxa0vivo murine alkali-burned cornea model was developed and treated with EGF and TRAM-34 eye drops to analyze the effect of these treatments on corneal healing and angiogenesis. The corneas were also analyzed by histological staining. The inxa0vitro results showed that EGF induces the upregulation of KCa3.1 and P-ERK in HUVECs and that this upregulation is suppressed by PD98059. EGF stimulates proliferation, migration and tube formation in HUVECs, and this effect can be suppressed by TRAM-34. TRAM-34 also arrests HUVECs in the G1 phase of the cell cycle and downregulates CDK4, vimentin and MMP-2 in these cells. The inxa0vivo results indicated that TRAM-34 suppresses EGF-induced corneal angiogenesis without affecting EGF-induced corneal wound healing. In summary, the upregulation of KCa3.1 may be crucial for EGF-induced angiogenesis through the MAPK/ERK signaling pathway. Thus, KCa3.1 may be a potential target for the treatment of EGF-induced corneal angiogenesis.

Triptolide Suppresses Alkali Burn-Induced Corneal Angiogenesis Along with a Downregulation of VEGFA and VEGFC Expression: TRIPTOLIDE SUPPRESSES CORNEAL ANGIOGENESIS

Triptolide (TPL) is an active compound extracted from a Chinese herbal medicine tripterygium wilfordii Hook. f. (Celastraceae), which has been used as an anti‐inflammatory drug for years. It also inhibits the growth and proliferation of different types of cancer cells. The inhibitory effect of TPL on angiogenesis after chemical‐induced corneal inflammation was studied in vivo. The effects of TPL on the proliferation, apoptosis, migration, and tube formation of rat aortic endothelial cells (RAECs) were studied in vitro. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. Migration was analyzed using the scratch wound healing assay and transwell assay. Tube formation assay was used to examine angiogenesis. Real‐time PCR and Western blot were used to determine the expression of vascular endothelial growth factor A (VEGFA) and VEGFC. To study the in vivo effects of TPL, the mouse model of alkali burn‐induced corneal angiogenesis was used. The angiogenesis was analyzed by determining the density of the newly generated blood vessels in corneas. We found that TPL induced apoptosis and inhibited the proliferation of RAECs in a dose‐dependent manner. TPL inhibited migration and tube formation of RAECs and decreased the expression of VEGFA and VEGFC in vitro. Furthermore, TPL suppressed alkali burn‐induced corneal angiogenesis and inhibited the expression of VEGFA and VEGFC in corneas in vivo. In conclusion, topical TPL as a pharmacological agent has the ability to reduce angiogenesis in cornea and may have clinical indications for the treatment of corneal angiogenesis diseases which have to be further explored. Anat Rec, 300:1348–1355, 2017.