Suppression of lymphangiogenesis in human lymphatic endothelial cells by simultaneously blocking VEGF-C and VEGF-D/VEGFR-3 with norcantharidin

Abstract

Lymph node metastasis of tumors is a crucial early step in the metastatic process. Tumor lymphangiogenesis plays an important role in promoting tumor metastasis to regional lymph nodes. Norcantharidin (NCTD) has been reported to possess potent anti-angiogenesis and antitumor properties in several cell lines and xenograft tumor models. However, its role in tumor-associated lymphangiogenesis and lymphatic metastasis remains unclear. Here, we investigated the effect of NCTD on proliferation, apoptosis, migration, invasion and the lymphatic tube formation, lymphangiogenesis, of human lymphatic endothelial cells (HLECs) in vitro by MTT, proliferation assay, Hoechst staining and flow cytometry, scraping line method, Matrigel invasion assay, inverted or fluorescence microscope and transmission electron microscope. Moreover, the underlying mechanisms, such as VEGF-C, VEGF-D, VEGFR-3 at protein and mRNA levels in lymphangiogenesis using 3-dimensional (3-D) culture of HLECs were measured by immunohistochemistry, western blotting and real-time polymerase chain reaction (RT-PCR). It was shown that NCTD inhibited proliferation, migration, invasion and lymphatic tube formation (forming-lymphatic and/or formed-lymphatic) of HLECs, induced HLEC apoptosis (all P<0.01) significantly, in a dose- and time-dependent manner (IC50 6.8 µg/ml); and downregulated the expression of VEGF-C, VEGF-D and VEGFR-3 at protein or/and mRNA levels (P<0.01) in HLEC lymphatic tube formation. Thus, we identified for the first time that NCTD inhibited HLEC lymphangiogenesis by simultaneously blocking VEGF-C and VEGF-D/VEGFR-3 in vitro. The present findings may be of importance to explore the therapeutical target or strategy of NCTD for tumor lymphangiogenesis and lymphatic metastasis.

Detection of CD133 (prominin‐1) in a human hepatoblastoma cell line (HuH‐6 clone 5)

“…Cells were cultured to near confluence at 37°C in 35‐mm culture dishes containing DMEM supplemented with 10% FBS. A linear scratch wound was made on the surface of the cultured cells using a sterile blade, according to the scratch wound healing assay (Liu et al, ). Then, the cells on one side of the scratch wound were completely scraped off using a plastic scraper, and the remaining cells were washed with Hank's buffered salt solution.…”

Vascular endothelial growth factor C expression is closely correlated with lymph node recurrence and poor prognosis in patients with early stage cervical cancer

“…VEGF-C has received attention as it is one of the most potent growth factors for inducing lymphangiogenesis and promoting lymph node metastasis in a wide range of human malignant tumours. 18,27 However, few data are available regarding the correlation between VEGF-C expression and tumour lymph node recurrence. 30 To our knowledge, the present clinical study is the first to detect VEGF-C mRNA expression in surgically resected pN0 cervical cancer tissues, and to investigate further the role of VEGF-C mRNA expression in relation to clinicopathological factors, lymph node recurrence and prognosis of this disease.…”

“…27 The VEGF family is the most widely investigated and most specific regulator of angiogenesis: it consists of six members: VEGF-A, -B, -C, -D, -E and placental growth factor. Among them, VEGF-C and -D are considered to be responsible for tumour lymphangiogenesis and lymph node metastasis.…”

Association between vascular endothelial growth factor expression and lymph node metastasis in cervical cancer: A meta‐analysis

“…Some studies have also shown that the expression of VEGF is significantly related with LNM in cervical cancer . However, the association between the expression of VEGF and LNM in cervical cancer has been reported in several studies, which obtained inconsistent conclusions . Hashimoto et al .…”