There is a pertinent need to develop prognostic biomarkers for practicing predictive, preventive and personalized medicine (PPPM) in colorectal cancer metastasis. The analysis of isoform expression data governed by alternative splicing provides a high-resolution picture of mRNAs in a defined condition. This information would not be available by studying gene expression changes alone. Hence, we utilized our prior data from an exon microarray and found ADAM12 and MUC4 to be strong biomarker candidates based on their alternative splicing scores and pattern. In this study, we characterized their isoform expression in a cell line model of metastatic colorectal cancer (SW480 & SW620). These two genes were found to be good prognostic indicators in two cohorts from The Cancer Genome Atlas database. We studied their exon structure using sequence information in the NCBI and ENSEMBL genome databases to amplify and validate six isoforms each for the ADAM12 and MUC4 genes. The differential expression of these isoforms was observed between normal, primary and metastatic colorectal cancer cell lines. RNA-Seq analysis further proved the differential expression of the gene isoforms. The isoforms of MUC4 and ADAM12 were found to change expression levels in response to 5-Fluorouracil (5-FU) treatment in a dose-, time- and cell line-dependent manner. Furthermore, we successfully detected the protein isoforms of ADAM12 and MUC4 in cell lysates, reflecting the differential expression at the protein level. The change in the mRNA and protein expression of MUC4 and ADAM12 in primary and metastatic cells and in response to 5-FU qualifies them to be studied as potential biomarkers. This comprehensive study underscores the importance of studying alternatively spliced isoforms and their potential use as prognostic and/or predictive biomarkers in the PPPM approach towards cancer.

The use of natural health products (NHP) is constantly increasing worldwide. Among the most NHP consumed are the resins of Commiphora myrrha (C. myrrha) tree. Consumption of C. myrrha may result in herb-drug interactions that can lead to serious health consequences. This study aims to determine the herb-drug interactions based on the induction of drug-metabolizing enzyme cytochrome P-450 (CYP) 2C9 by a non-toxic boiled aqueous extract of C. myrrha and to unveil the potential involvement of xenobiotic-sensing nuclear receptors such as Pregnane X Receptor (PXR), a well-known CYP 2C9 transcription factor. Tested at low concentrations (0.01-10 g/mL) devoid of anti-proliferative effects, boiled C. myrrha resin aqueous extract upregulated CYP 2C9 gene and protein expressions in a dose-dependent manner, reaching a mean expression level exceeding 3.0-fold change in Hep G2 cells, compared with the basal level expressed in untreated cells. Furthermore, chemical analysis using mass spectrometry and computational molecular docking using in silico studies revealed that few C. myrrha resin-derived metabolites are strongly bound to PXR. In particular, the identified metabolite named diayangambin exhibited a predicted molecular docking score close to that of a native PXR ligand. The use of the nuclear receptor binding assays confirmed the binding between C. myrrha metabolite(s) and PXR, while no binding to the other xenobiotic-sensing nuclear receptor, the constitutive androstane receptor (CAR), was observed, suggesting that C. myrrha metabolite(s) are potential PXR agonists. In conclusion, although in vivo studies are needed, this study cautions patients, healthcare providers, and governmental regulators against the consumption of C. myrrha and diayangambin-rich preparations with a high potential for herb-drug interactions through drug-metabolizing CYP 2C9 enzyme induction, which could affect drug efficacy.