Melissa R. Ingala
Vascular endothelial growth factor (VEGFA) is a eukaryotic growth factor which normally regulates angiogenesis during embryonic development and following exercise or injury . Pathological, VEGFA- stimulated angiogenesis is involved in the metastasis of solid tumors as well as the development of age-related macular degeneration . Tumor cells overcome hypoxia by secreting VEGFA in an autocrine loop, leading to neovascularization and subsequent oxygen delivery . In the macula of the retina, an imbalance of VEGFA with its antagonistic regulator PEDF leads to choroid neovascularization, hemorrhaging, and scarring of retinal tissue .
A number of VEGFA isoforms have been described. The VEGFA gene contains eight exons which encode the functional domains of the protein [Fig 1.]. Isoforms are generated by alternative splicing, post-translational modification, and stop-codon read-through mechanisms [5,6]. There is evidence that the angiogenic potential of VEGFA isoforms is dependent in large part upon which splice form is produced . The smallest isoforms [15-17 kDa] lack exons six and seven, are diffusible, and thus have the potential to cause metastasis; full-length isoforms are immobile because they are associated with the extracellular matrix [8,9].
It has been demonstrated previously that chemical compounds can change alternative splicing in cancer cells to favor one splice variant over another [10,11]. More recently, it has been shown that nutraceuticals, or pharmacologically relevant compounds ingested in the diet, can also affect alternative splicing in other transcripts . Therefore, the research question for this experiment was: can nutraceuticals which are known to alter splicing also affect VEGFA splicing?
Materials and Methods
Immortalized colorectal cancer cells (Caco-2) were treated with a variety of nutraceutical agents. Controls in all experiments were untreated Caco-2 cells grown at the same time and under the same conditions as treated groups.
To determine if nutraceuticals impact alternative splicing, RNA was purified from treated and untreated cells using the RNeasy® Plus Mini Kit (QIAGEN, Germany). Intron-spanning primer pairs were developed to span exons four through the 3’ UTR just after exon eight [Fig. 2]. RT-PCR was performed using a One-Step RT-PCR kit per the manufacturer’s instructions (QIAGEN, Germany). PCR products were run on a 1% agarose gel and visualized with ethidium bromide on a UV illuminator (Carestream, USA). Band intensities for each treatment were quantified and compared using ImageJ (NIH, Bethesda, MD, USA). PCR products were purified using the QIAQuick Gel Extraction Kit (QIAGEN, Germany) and sent for sequencing (GENEWIZ, NJ, USA). Recovered sequences were BLASTed against NCBI’s database, and alternative transcripts were identified.
To test for effects at the protein level, the same nutraceutical treatments were applied to new Caco-2 cells. Cytosolic proteins were extracted from treated and untreated cells and were run on a 10% Bis-Tris SDS-PAGE gel (Life Technologies, MA, USA). Proteins were transferred to nitrocellulose membrane and probed using a polyclonal rabbit anti-VEGFA antibody (Abcam, Cambridge, UK). Protein isoforms were recorded.
Results of the RT-PCR experiments show two prominent splice variants of 570 bp and 430 bp [Fig 3a]. BLAST results of sequences corresponding to these bands indicate that the 570 bp band matches a known VEGFA variant which lacks exon six (Δ6). The 430 bp band matches a known variant which lacks both exons six and seven (Δ6,7) [Fig 3b]. Untreated cells produced bands such that the Δ6 band was more intense than the Δ6,7 band. This pattern was also observed for all nutraceutical-treated groups except for #54. The #54 treatment produced a trend such that the Δ6,7 band was more intense than the Δ6 band. Band intensities were thus quantified in ImageJ and presented as a ratio of Δ6:Δ6,7. Indeed, #54 produced a ratio of 0.67, indicating that the Δ6,7 band was more intense than the Δ6 band for that treatment [Fig 3c]. The predicted molecular weight of the Δ6 isoform is 40.7 kDa, while the molecular weight for the Δ6,7 isoform is variable (17-35 kDa) due to the possibility of other splicing events.
At the protein level, an immunoblot confirmed that treatment #54 produced less of a 40 kDa isoform than untreated control [Fig. 4]. To confirm that this result was not the product of toxicity or imprecise loading, the blot was stained with Coomassie Brilliant Blue [data not shown]. The resulting stain showed that all lanes were loaded with the same amount of protein. Therefore, it was confirmed that treatment #54 reduced the amount of the 40 kDa, Δ6 isoform. The Δ6,7 isoform was not identified on the immunoblot of cytosolic proteins; this isoform is not cell-associated, and therefore, as expected, diffused into the conditioned medium.
This experiment presents evidence that nutraceutical treatment affects alternative splicing of VEGFA. Treatment with #54 produces relatively more of the Δ6,7 isoform than the Δ6 compared with untreated control. The most common VEGFA isoform is the one which lacks exon 6, but includes seven . This isoform is primarily cell-associated, because exon seven codes for a neuropilin [ECM receptor] binding domain . The isoform whose production is increased by #54 lacks both exons six and seven; this isoform is diffusible, and thus has the potential to create metastases in other tissues . Currently, physicians prescribe bevacizumab to treat metastatic colorectal cancer; this treatment relies on an anti-VEGFA antibody which sequesters diffusible VEGFA . A nutraceutical which can reduce endogenous expression of diffusible VEGFA would therefore augment current treatments. Future studies should include larger screens of nutraceuticals to identify any which increase the proportion of cell-associated VEGFA isoforms.
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