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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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Quantification of mRNA expression of pan-VEGF and VEGFxxx isoform mRNA by Q-PCR. (A and B) Primers that detected all isoforms were used to detect increasing amounts of VEGF165b (A) or VEGF165 (B). (C) Standard curves for the two isoforms were the same indicating that a mixture of both could be assessed equally. (D) Amplification of VEGFxxx cDNA specifically. Primers specific to exon 8a resulted in amplification that was more than six orders of magnitude more sensitive for VEGF165 than VEGF165b (dotted line). (E) Standard curve using VEGF165 as a template.
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fig1: Quantification of mRNA expression of pan-VEGF and VEGFxxx isoform mRNA by Q-PCR. (A and B) Primers that detected all isoforms were used to detect increasing amounts of VEGF165b (A) or VEGF165 (B). (C) Standard curves for the two isoforms were the same indicating that a mixture of both could be assessed equally. (D) Amplification of VEGFxxx cDNA specifically. Primers specific to exon 8a resulted in amplification that was more than six orders of magnitude more sensitive for VEGF165 than VEGF165b (dotted line). (E) Standard curve using VEGF165 as a template.

Mentions: Figures 1A and B show examples of the reverse transcription-polymerase chain reaction (RT-PCR) curves for VEGF165b and VEGF165 templates respectively using pan-VEGF primers. Figure 1C shows the standard curve generated from cycle threshold for the two templates, showing that there was no difference in the standard curves (n=3). Thus the efficiency of amplification of the two templates is not different. Figure 1D shows the amplification curves for the exon 8a primers using the VEGF165 sequence as a template (VEGF165b template did not result in amplification until 18 cycles later than equivalent VEGF165 concentration). Total VEGF and VEGFxxx copy numbers were calculated for each sample using the calibration curve shown in Figure 1E. The difference between the total VEGF and the VEGFxxx copy number was assumed to be the VEGF165b copy number.


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)

Quantification of mRNA expression of pan-VEGF and VEGFxxx isoform mRNA by Q-PCR. (A and B) Primers that detected all isoforms were used to detect increasing amounts of VEGF165b (A) or VEGF165 (B). (C) Standard curves for the two isoforms were the same indicating that a mixture of both could be assessed equally. (D) Amplification of VEGFxxx cDNA specifically. Primers specific to exon 8a resulted in amplification that was more than six orders of magnitude more sensitive for VEGF165 than VEGF165b (dotted line). (E) Standard curve using VEGF165 as a template.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2361696&req=5

fig1: Quantification of mRNA expression of pan-VEGF and VEGFxxx isoform mRNA by Q-PCR. (A and B) Primers that detected all isoforms were used to detect increasing amounts of VEGF165b (A) or VEGF165 (B). (C) Standard curves for the two isoforms were the same indicating that a mixture of both could be assessed equally. (D) Amplification of VEGFxxx cDNA specifically. Primers specific to exon 8a resulted in amplification that was more than six orders of magnitude more sensitive for VEGF165 than VEGF165b (dotted line). (E) Standard curve using VEGF165 as a template.
Mentions: Figures 1A and B show examples of the reverse transcription-polymerase chain reaction (RT-PCR) curves for VEGF165b and VEGF165 templates respectively using pan-VEGF primers. Figure 1C shows the standard curve generated from cycle threshold for the two templates, showing that there was no difference in the standard curves (n=3). Thus the efficiency of amplification of the two templates is not different. Figure 1D shows the amplification curves for the exon 8a primers using the VEGF165 sequence as a template (VEGF165b template did not result in amplification until 18 cycles later than equivalent VEGF165 concentration). Total VEGF and VEGFxxx copy numbers were calculated for each sample using the calibration curve shown in Figure 1E. The difference between the total VEGF and the VEGFxxx copy number was assumed to be the VEGF165b copy number.

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