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VEGF 165 b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy.

Varey AH, Rennel ES, Qiu Y, Bevan HS, Perrin RM, Raffy S, Dixon AR, Paraskeva C, Zaccheo O, Hassan AB, Harper SJ, Bates DO - Br. J. Cancer (2008)

Bottom Line: However, although bevacizumab effectively inhibited the rapid growth of colon carcinomas expressing VEGF(165), it did not affect the slower growth of tumours from colonic carcinoma cells expressing VEGF(165)b.These results show that the balance of antiangiogenic to proangiogenic isoforms switches to a variable extent in CRC, regulates tumour growth rates and affects the sensitivity of tumours to bevacizumab by competitive binding.Together with the identification of an autocrine cytoprotective role for VEGF(165)b in colonic epithelial cells, these results indicate that bevacizumab treatment of human CRC may depend upon this balance of VEGF isoforms.

View Article: PubMed Central - PubMed

Affiliation: Microvascular Research Laboratories, Department of Physiology and Pharmacology, School of Veterinary Sciences, University of Bristol, Bristol, UK.

ABSTRACT
Bevacizumab, an anti-vascular endothelial growth factor (VEGF-A) antibody, is used in metastatic colorectal carcinoma (CRC) treatment, but responses are unpredictable. Vascular endothelial growth factor is alternatively spliced to form proangiogenic VEGF(165) and antiangiogenic VEGF(165)b. Using isoform-specific enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, we found that over 90% of the VEGF in normal colonic tissue was VEGF(xxx)b, but there was a variable upregulation of VEGF(xxx) and downregulation of VEGF(xxx)b in paired human CRC samples. Furthermore, cultured colonic adenoma cells expressed predominantly VEGF(xxx)b, whereas colonic carcinoma cells expressed predominantly VEGF(xxx). However, adenoma cells exposed to hypoxia switched their expression from predominantly VEGF(xxx)b to predominantly VEGF(xxx). VEGF(165)b overexpression in LS174t colon cancer cells inhibited colon carcinoma growth in mouse xenograft models. Western blotting and surface plasmon resonance showed that VEGF(165)b bound to bevacizumab with similar affinity as VEGF(165). However, although bevacizumab effectively inhibited the rapid growth of colon carcinomas expressing VEGF(165), it did not affect the slower growth of tumours from colonic carcinoma cells expressing VEGF(165)b. Both bevacizumab and anti-VEGF(165)b-specific antibodies were cytotoxic to colonic epithelial cells, but less so to colonic carcinoma cells. These results show that the balance of antiangiogenic to proangiogenic isoforms switches to a variable extent in CRC, regulates tumour growth rates and affects the sensitivity of tumours to bevacizumab by competitive binding. Together with the identification of an autocrine cytoprotective role for VEGF(165)b in colonic epithelial cells, these results indicate that bevacizumab treatment of human CRC may depend upon this balance of VEGF isoforms.

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VEGF165b mRNA is expressed in human colon tissue and colon cancer. (A) VEGFxxxb mRNA is expressed in normal and cancerous colon. PCR of cDNA reverse transcribed from RNA extracted from paired human colon samples shows two bands, the proximal splice isoforms (VEGFxxx, ∼200 bp) and the distal splice isoforms (VEGFxxxb, ∼135 bp). (B–D) Q-PCR for pan-VEGF (VEGF165b and VEGF165) and VEGF165 isoforms. (B) Primers that detected all 165 amino acid-coding isoforms were used to detect increasing amounts of total VEGF (VEGF165b and VEGF165). (C) Exon 8a-specific primers were used to measure the amount of VEGF165, which was significantly increased in colon cancers, P<0.01. (D) VEGF165b levels calculated from the VEGF165 and total VEGF levels.
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fig4: VEGF165b mRNA is expressed in human colon tissue and colon cancer. (A) VEGFxxxb mRNA is expressed in normal and cancerous colon. PCR of cDNA reverse transcribed from RNA extracted from paired human colon samples shows two bands, the proximal splice isoforms (VEGFxxx, ∼200 bp) and the distal splice isoforms (VEGFxxxb, ∼135 bp). (B–D) Q-PCR for pan-VEGF (VEGF165b and VEGF165) and VEGF165 isoforms. (B) Primers that detected all 165 amino acid-coding isoforms were used to detect increasing amounts of total VEGF (VEGF165b and VEGF165). (C) Exon 8a-specific primers were used to measure the amount of VEGF165, which was significantly increased in colon cancers, P<0.01. (D) VEGF165b levels calculated from the VEGF165 and total VEGF levels.

Mentions: To determine whether VEGF165b and VEGF165 mRNA were expressed in normal and cancerous colon, RT-PCR using primers that distinguish between the two families of isoforms was carried out on eight pairs of samples. Reverse transcription-polymerase chain reaction gave two bands, one at ∼135 bp, consistent with VEGF165b or VEGF189b, and one at ∼200 bp, consistent with VEGF165 and VEGF189. This size difference was due to the splicing out of exon 8a in the VEGFxxxb family, resulting in the shorter mRNA (although exon 8b is present in the mRNA of the VEGFxxx family, a stop codon in exon 8a prevents its translation). VEGFxxx and VEGFxxxb mRNA expression was detected in both normal and tumour tissue (Figure 4A).


VEGF 165 b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy.

Varey AH, Rennel ES, Qiu Y, Bevan HS, Perrin RM, Raffy S, Dixon AR, Paraskeva C, Zaccheo O, Hassan AB, Harper SJ, Bates DO - Br. J. Cancer (2008)

VEGF165b mRNA is expressed in human colon tissue and colon cancer. (A) VEGFxxxb mRNA is expressed in normal and cancerous colon. PCR of cDNA reverse transcribed from RNA extracted from paired human colon samples shows two bands, the proximal splice isoforms (VEGFxxx, ∼200 bp) and the distal splice isoforms (VEGFxxxb, ∼135 bp). (B–D) Q-PCR for pan-VEGF (VEGF165b and VEGF165) and VEGF165 isoforms. (B) Primers that detected all 165 amino acid-coding isoforms were used to detect increasing amounts of total VEGF (VEGF165b and VEGF165). (C) Exon 8a-specific primers were used to measure the amount of VEGF165, which was significantly increased in colon cancers, P<0.01. (D) VEGF165b levels calculated from the VEGF165 and total VEGF levels.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2361696&req=5

fig4: VEGF165b mRNA is expressed in human colon tissue and colon cancer. (A) VEGFxxxb mRNA is expressed in normal and cancerous colon. PCR of cDNA reverse transcribed from RNA extracted from paired human colon samples shows two bands, the proximal splice isoforms (VEGFxxx, ∼200 bp) and the distal splice isoforms (VEGFxxxb, ∼135 bp). (B–D) Q-PCR for pan-VEGF (VEGF165b and VEGF165) and VEGF165 isoforms. (B) Primers that detected all 165 amino acid-coding isoforms were used to detect increasing amounts of total VEGF (VEGF165b and VEGF165). (C) Exon 8a-specific primers were used to measure the amount of VEGF165, which was significantly increased in colon cancers, P<0.01. (D) VEGF165b levels calculated from the VEGF165 and total VEGF levels.
Mentions: To determine whether VEGF165b and VEGF165 mRNA were expressed in normal and cancerous colon, RT-PCR using primers that distinguish between the two families of isoforms was carried out on eight pairs of samples. Reverse transcription-polymerase chain reaction gave two bands, one at ∼135 bp, consistent with VEGF165b or VEGF189b, and one at ∼200 bp, consistent with VEGF165 and VEGF189. This size difference was due to the splicing out of exon 8a in the VEGFxxxb family, resulting in the shorter mRNA (although exon 8b is present in the mRNA of the VEGFxxx family, a stop codon in exon 8a prevents its translation). VEGFxxx and VEGFxxxb mRNA expression was detected in both normal and tumour tissue (Figure 4A).

Bottom Line: However, although bevacizumab effectively inhibited the rapid growth of colon carcinomas expressing VEGF(165), it did not affect the slower growth of tumours from colonic carcinoma cells expressing VEGF(165)b.These results show that the balance of antiangiogenic to proangiogenic isoforms switches to a variable extent in CRC, regulates tumour growth rates and affects the sensitivity of tumours to bevacizumab by competitive binding.Together with the identification of an autocrine cytoprotective role for VEGF(165)b in colonic epithelial cells, these results indicate that bevacizumab treatment of human CRC may depend upon this balance of VEGF isoforms.

View Article: PubMed Central - PubMed

Affiliation: Microvascular Research Laboratories, Department of Physiology and Pharmacology, School of Veterinary Sciences, University of Bristol, Bristol, UK.

ABSTRACT
Bevacizumab, an anti-vascular endothelial growth factor (VEGF-A) antibody, is used in metastatic colorectal carcinoma (CRC) treatment, but responses are unpredictable. Vascular endothelial growth factor is alternatively spliced to form proangiogenic VEGF(165) and antiangiogenic VEGF(165)b. Using isoform-specific enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, we found that over 90% of the VEGF in normal colonic tissue was VEGF(xxx)b, but there was a variable upregulation of VEGF(xxx) and downregulation of VEGF(xxx)b in paired human CRC samples. Furthermore, cultured colonic adenoma cells expressed predominantly VEGF(xxx)b, whereas colonic carcinoma cells expressed predominantly VEGF(xxx). However, adenoma cells exposed to hypoxia switched their expression from predominantly VEGF(xxx)b to predominantly VEGF(xxx). VEGF(165)b overexpression in LS174t colon cancer cells inhibited colon carcinoma growth in mouse xenograft models. Western blotting and surface plasmon resonance showed that VEGF(165)b bound to bevacizumab with similar affinity as VEGF(165). However, although bevacizumab effectively inhibited the rapid growth of colon carcinomas expressing VEGF(165), it did not affect the slower growth of tumours from colonic carcinoma cells expressing VEGF(165)b. Both bevacizumab and anti-VEGF(165)b-specific antibodies were cytotoxic to colonic epithelial cells, but less so to colonic carcinoma cells. These results show that the balance of antiangiogenic to proangiogenic isoforms switches to a variable extent in CRC, regulates tumour growth rates and affects the sensitivity of tumours to bevacizumab by competitive binding. Together with the identification of an autocrine cytoprotective role for VEGF(165)b in colonic epithelial cells, these results indicate that bevacizumab treatment of human CRC may depend upon this balance of VEGF isoforms.

Show MeSH
Related in: MedlinePlus