VEGF111b, a new member of VEGFxxxb isoforms and induced by mitomycin C, inhibits angiogenesis

https://doi.org/10.1016/j.bbrc.2013.09.144Get rights and content

Highlights

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    We discovered a new member of VEGFxxxb family-VEGF111b.

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    We found VEGF111b mRNA and protein can be induced by mitomycin C.

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    We confirmed VEGF111b over-expression inhibits angiogenesis.

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    VEGF111b inhibits angiogenesis through inhibiting VEGF-R2/PI3K/Akt and VEGF-R2/ERK1/2 phosphorylation.

Abstract

Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA3.1 empty vector, pcDNA3.1-VEGF111b or pcDNA3.1-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth.

Introduction

Angiogenesis plays a key role in tumor growth and progression [1]. A principal angiogenic promoter that stimulates the migration of endothelial cells, sprouting of blood vessels, and generation of new vessels from existing vascular endothelium in tumors is the vascular endothelial growth factor (VEGF-A) [2], [3]. Anti-angiogenic therapy targeting VEGF-A is becoming an additional therapeutic strategy to surgery, chemotherapy and radiotherapy, which has attracted more attention.

The human VEGF-A gene has been assigned to chromosome 6p21.3. It contains 8 exons, separated by seven introns, and its coding region spans approximately 14 kb [4]. Alternative splicing of full-length VEGF pre-mRNA gives rise to two known families of protein isoforms that differ by only six amino acids at their C-terminal end (Fig. 1). The conventional VEGFxxx isoforms, where xxx refers to the number of amino acids, are formed by the proximal splice site (PSS) selection in exon 8 (termed exon 8a) and differentially splicing in exons 5, 6 or 7 [5]. The six amino acids encoded by exon 8a are CDKPRR (Fig. 1). The major isoforms of VEGFxxx family, demonstrated to be pro-angiogenic, are VEGF165, VEGF189, VEGF121, VEGF145, VEGF183, VEGF206 and VEGF111 [6]. However, another sister family of VEGF isoforms, generically referred to as VEGFxxxb isoforms, are formed by distal splice site (DSS) selection 66 bp downstream of the PSS site in exon 8 (termed exon 8b) [7], [8], [9]. Exon 8b encodes a unique amino acids sequence SLTRKD (Fig. 1). In VEGFxxxb family, VEGF165b, VEGF121b, VEGF145b and VEGF183b have been identified in succession and demonstrated to be anti-angiogenic [5]. The first verified and widely reported VEGFxxxb family member is VEGF165b, which has been clearly shown to inhibit endothelial cell growth and migration in vitro and angiogenesis in tumor and non-tumor-related angiogenesis [7], [10], [11].

Although a large number of evidences on the expression and property of VEGF165b have already been published, there is very little evidence on other VEGFxxxb family members, and their existence and property is still unknown. In 2007, Mineur reported a new VEGFxxx family member, VEGF111, and demonstrated that it could only be induced in the condition of genotoxic agents, such as camptothecin, mimosin, mitomycin C and UV-B [12]. The VEGF111 coding sequence consists of exons 1–4 and 8a. DSS in exon 8 has stronger splicing advantages than PSS [13]. Whether VEGF111b exits and plays a role in anti-angiogenic effect has never been demonstrated. Thus we speculate the presence of VEGF111b. Therefore, in this study, we detected and discovered a new member of VEGFxxxb family, VEGF111b, under the induction of mitomycin C. We constructed eukaryotic expression vector of VEGF111b for sequencing, prepared VEGF111b polyclonal antibody, and finally confirmed the hypothesis that VEGF111b also show anti-angiogenic properties.

Section snippets

Reagents and antibodies

Mitomycin C was obtained from Sigma–Aldrich (Saint Quentin Fallavier, France). Anti-VEGF-R1, and anti-VEGF-R2 were purchased from Beyotime (Jiangsu, China). All other primary antibodies were purchased from Abcam (Cambridge, TX, USA). Horseradish peroxidase (HRP)-labeled anti-mouse and anti-rabbit secondary antibodies were from Santa Cruz (Dallas, TX, USA).

Cell lines

Human umbilical vein endothelial cells (HUVECs) were extracted from umbilical cords from caesarean sections (The General Hospital of the

VEGF111b expression could be induced by mitomycin C in SKOV3 cells

According to alternative splicing of VEGFxxx and VEGFxxxb families, We designed VEGF111b specific primers and observed VEGF111b mRNA production (Fig. 1A, lane 1) by using RT-PCR in SKOV3 cells after treatment with 100 μg/ml mitomycin C. Sequence analysis of VEGF111b were shown in Supplementary Fig. S1 and demonstrated that VEGF111b is composed of exons 1–4 and 8b. This product was not observed in mitomycin C-unexposed cells (Fig. 1A, lane 2). Theoretically this new splice variant encodes a 111

Discussion

Here, we list new data for the first time presenting existence of VEGF111b as a different VEGFxxxb family member. Similar to VEGF111 [12], the mRNA and protein expression of VEGF111b are induced in the human ovarian cancer cells by genotoxic agents such as mitomycin C. We failed to detect VEGF111b without mitomycin C treatment. Thus, the expression of VEGF111b similarly depends upon the treatment of mitomycin C inducing double-strand breaks. Its expression in other types of cultured cells, or

Author contributions

G.F. and L.X.L. conceived and designed the study. G.F. and J.K. carried out the experimental work and wrote the manuscript. M.S. and B.P. interpreted the data. Y.Y.Q. and L.M.Z. conceived and designed the study and wrote the manuscript.

Sources of funding

This work was supported by Grants 2010CB912504 and 2011CB503900 from “973” National S&T Major Project, 81250030, 81170101, 81370235, and 81322005 from the National Natural Science Foundation of China, and 7122106 from the Natural Science Foundation of Beijing, China.

Disclosures

The authors disclosed no potential conflicts of interest.

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    These authors contributed equally to this work.

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